Cysteine engineered antibodies and conjugates

ABSTRACT

Antibodies are engineered by replacing one or more amino acids of a parent antibody with non cross-linked, highly reactive cysteine amino acids. Antibody fragments may also be engineered with one or more cysteine amino acids to form cysteine engineered antibody fragments (ThioFab). Methods of design, preparation, screening, and selection of the cysteine engineered antibodies are provided. Cysteine engineered antibodies (Ab), optionally with an albumin-binding peptide (ABP) sequence, are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered antibody-drug conjugates having Formula I: 
       Ab-(L-D) p   I 
     where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.

This application is a divisional of U.S. Ser. No. 11/233,258 filed onSep. 22, 2005, now U.S. Pat. No. 7,521,541 issued Apr. 21, 2009, andalso claims the benefit of priority under 35 USC §119(e) of U.S.Provisional Application Ser. No. 60/612,468 filed on Sep. 23, 2004 andU.S. Provisional Application Ser. No. 60/696,353 filed on Jun. 30, 2005,each of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to antibodies engineered with reactivecysteine residues and more specifically to antibodies with therapeuticor diagnostic applications. The cysteine engineered antibodies may beconjugated with chemotherapeutic drugs, toxins, affinity ligands such asbiotin, and detection labels such as fluorophores. The invention alsorelates to methods of using antibodies and antibody-drug conjugatecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders. Inattempts to discover effective cellular targets for cancer diagnosis andtherapy with antibodies, researchers have sought to identifytransmembrane or otherwise tumor-associated polypeptides that arespecifically expressed on the surface of cancer cells as compared tonormal, non-cancerous cell(s). The identification of suchtumor-associated cell surface antigen polypeptides, i.e. tumorassociated antigens (TAA), has given rise to the ability to specificallytarget cancer cells for destruction via antibody-based therapies.

The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, forthe local delivery of cytotoxic or cytostatic agents, i.e. drugs to killor inhibit tumor cells in the treatment of cancer (Lambert, J. (2005)Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) NatureBiotechnology 23(9):1137-1146; Payne, G. (2003) Cancer Cell 3:207-212;Syrigos and Epenetos (1999) Anticancer Research 19:605-614;Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S.Pat. No. 4,975,278) theoretically allows targeted delivery of the drugmoiety to tumors, and intracellular accumulation therein, where systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15,1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, A. Pinchera et al (ed.s), pp. 475-506). Maximalefficacy with minimal toxicity is sought thereby. Efforts to design andrefine ADC have focused on the selectivity of monoclonal antibodies(mAbs) as well as drug-linking and drug-releasing properties (Lambert,J. (2005) Curr. Opinion in Pharmacology 5:543-549. Both polyclonalantibodies and monoclonal antibodies have been reported as useful inthese strategies (Rowland et al (1986) Cancer Immunol. Immunother.,21:183-87). Drugs used in these methods include daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al (1986) supra). Toxins used inantibody-toxin conjugates include bacterial toxins such as diphtheriatoxin, plant toxins such as ricin, small molecule toxins such asgeldanamycin (Mandler et al (2000) J. of the Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;Hinman et al (1993) Cancer Res. 53:3336-3342). The toxins may effecttheir cytotoxic and cytostatic effects by mechanisms including tubulinbinding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugstend to be inactive or less active when conjugated to large antibodiesor protein receptor ligands.

An antibody-radioisotope conjugate has been approved. ZEVALIN®(ibritumomab tiuxetan, Biogen/Idec) is composed of a murine IgG1 kappamonoclonal antibody directed against the CD20 antigen found on thesurface of normal and malignant B lymphocytes and ¹¹¹In or ⁹⁰Yradioisotope bound by a thiourea linker-chelator (Wiseman et al (2000)Eur. J. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood99(12):4336-42; Witzig et al (2002) J. Clin. Oncol. 20(10):2453-63;Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69). Although ZEVALIN®has activity against B-cell non-Hodgkin's Lymphoma (NHL), administrationresults in severe and prolonged cytopenias in most patients. MYLOTARG™(gemtuzumab ozogamicin, Wyeth Pharmaceuticals), an antibody-drugconjugate composed of a hu CD33 antibody linked to calicheamicin, wasapproved in 2000 for the treatment of acute myeloid leukemia byinjection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), anantibody-drug conjugate composed of the huC242 antibody linked via thedisulfide linker SPP to the maytansinoid drug moiety, DM1 (Xie et al(2004) J. of Pharm. and Exp. Ther. 308(3):1073-1082), is advancing intoPhase TI trials for the treatment of cancers that express CanAg, such ascolon, pancreatic, gastric, and others. MLN-2704 (Millennium Pharm., BZLBiologics, Immunogen Inc.), an antibody-drug conjugate composed of theanti-prostate specific membrane antigen (PSMA) monoclonal antibodylinked to the maytansinoid drug moiety, DM1, is under development forthe potential treatment of prostate tumors.

The auristatin peptides, auristatin E (AE) and monomethylauristatin(MMAE), synthetic analogs of dolastatin (WO 02/088172), have beenconjugated to: (i) chimeric monoclonal antibodies cBR96 (specific toLewis Y on carcinomas); (ii) cAC10 which is specific to CD30 onhematological malignancies (Klussman, et al (2004), BioconjugateChemistry 15(4):765-773; Doronina et al (2003) Nature Biotechnology21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465; US2004/0018194; (iii) anti-CD20 antibodies such as rituxan (WO 04/032828)for the treatment of CD20-expressing cancers and immune disorders; (iv)anti-EphB2R antibodies 2H9 and anti-IL-8 for treatment of colorectalcancer (Mao et al (2004) Cancer Research 64(3):781-788); (v) E-selectinantibody (Bhaskar et al (2003) Cancer Res. 63:6387-6394); and (vi) otheranti-CD30 antibodies (WO 03/043583). Variants of auristatin E aredisclosed in U.S. Pat. No. 5,767,237 and U.S. Pat. No. 6,124,431.Monomethyl auristatin E conjugated to monoclonal antibodies aredisclosed in Senter et al, Proceedings of the American Association forCancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004. Auristatin analogs MMAE and MMAF have been conjugated to variousantibodies (WO 2005/081711).

Conventional means of attaching, i.e. linking through covalent bonds, adrug moiety to an antibody generally leads to a heterogeneous mixture ofmolecules where the drug moieties are attached at a number of sites onthe antibody. For example, cytotoxic drugs have typically beenconjugated to antibodies through the often-numerous lysine residues ofan antibody, generating a heterogeneous antibody-drug conjugate mixture.Depending on reaction conditions, the heterogeneous mixture typicallycontains a distribution of antibodies with from 0 to about 8, or more,attached drug moieties. In addition, within each subgroup of conjugateswith a particular integer ratio of drug moieties to antibody, is apotentially heterogeneous mixture where the drug moiety is attached atvarious sites on the antibody. Analytical and preparative methods areinadequate to separate and characterize the antibody-drug conjugatespecies molecules within the heterogeneous mixture resulting from aconjugation reaction. Antibodies are large, complex and structurallydiverse biomolecules, often with many reactive functional groups. Theirreactivities with linker reagents and drug-linker intermediates aredependent on factors such as pH, concentration, salt concentration, andco-solvents. Furthermore, the multistep conjugation process may benonreproducible due to difficulties in controlling the reactionconditions and characterizing reactants and intermediates.

Cysteine thiols are reactive at neutral pH, unlike most amines which areprotonated and less nucleophilic near pH 7. Since free thiol (RSH,sulfhydryl) groups are relatively reactive, proteins with cysteineresidues often exist in their oxidized form as disulfide-linkedoligomers or have internally bridged disulfide groups. Extracellularproteins generally do not have free thiols (Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London,at page 55). The amount of free thiol in a protein may be estimated bythe standard Ellman's assay. Immunoglobulin M is an example of adisulfide-linked pentamer, while immunoglobulin G is an example of aprotein with internal disulfide bridges bonding the subunits together.In proteins such as this, reduction of the disulfide bonds with areagent such as dithiothreitol (DTT) or selenol (Singh et al (2002)Anal. Biochem. 304:147-156) is required to generate the reactive freethiol. This approach may result in loss of antibody tertiary structureand antigen binding specificity.

Antibody cysteine thiol groups are generally more reactive, i.e. morenucleophilic, towards electrophilic conjugation reagents than antibodyamine or hydroxyl groups. Cysteine residues have been introduced intoproteins by genetic engineering techniques to form covalent attachmentsto ligands or to form new intramolecular disulfide bonds (Better et al(1994) J. Biol. Chem. 269(13):9644-9650; Bernhard et al (1994)Bioconjugate Chem. 5:126-132; Greenwood et al (1994) TherapeuticImmunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci. USA96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214; Chmuraet al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No.6,248,564). However, designing in cysteine thiol groups by the mutationof various amino acid residues of a protein to cysteine amino acids ispotentially problematic, particularly in the case of unpaired (free Cys)residues or those which are relatively accessible for reaction oroxidation. In concentrated solutions of the protein, whether in theperiplasm of E. coli, culture supernatants, or partially or completelypurified protein, unpaired Cys residues on the surface of the proteincan pair and oxidize to form intermolecular disulfides, and henceprotein dimers or multimers. Disulfide dimer formation renders the newCys unreactive for conjugation to a drug, ligand, or other label.Furthermore, if the protein oxidatively forms an intramoleculardisulfide bond between the newly engineered Cys and an existing Cysresidue, both Cys groups are unavailable for active site participationand interactions. Furthermore, the protein may be rendered inactive ornon-specific, by misfolding or loss of tertiary structure (Zhang et al(2002) Anal. Biochem. 311:1-9).

SUMMARY

The compounds of the invention include cysteine engineered antibodieswhere one or more amino acids of a parent antibody are replaced with afree cysteine amino acid. A cysteine engineered antibody comprises oneor more free cysteine amino acids having a thiol reactivity value in therange of 0.6 to 1.0. A free cysteine amino acid is a cysteine residuewhich has been engineered into the parent antibody and is not part of adisulfide bridge.

In one aspect, the cysteine engineered antibody is prepared by a processcomprising:

-   -   (a) replacing one or more amino acid residues of a parent        antibody by cysteine; and    -   (b) determining the thiol reactivity of the cysteine engineered        antibody by reacting the cysteine engineered antibody with a        thiol-reactive reagent.

The cysteine engineered antibody may be more reactive than the parentantibody with the thiol-reactive reagent.

The free cysteine amino acid residues may be located in the heavy orlight chains, or in the constant or variable domains. Antibodyfragments, e.g. Fab, may also be engineered with one or more cysteineamino acids replacing amino acids of the antibody fragment, to formcysteine engineered antibody fragments.

Another aspect of the invention provides a method of preparing (making)a cysteine engineered antibody, comprising:

-   -   (a) introducing one or more cysteine amino acids into a parent        antibody in order to generate the cysteine engineered antibody;        and    -   (b) determining the thiol reactivity of the cysteine engineered        antibody with a thiol-reactive reagent;

wherein the cysteine engineered antibody is more reactive than theparent antibody with the thiol-reactive reagent.

Step (a) of the method of preparing a cysteine engineered antibody maycomprise:

-   -   (i) mutagenizing a nucleic acid sequence encoding the cysteine        engineered antibody;    -   (ii) expressing the cysteine engineered antibody; and    -   (iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of preparing a cysteine engineered antibody maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of preparing a cysteine engineered antibody mayalso comprise:

-   -   (i) reacting the cysteine engineered antibody with a        thiol-reactive affinity reagent to generate an affinity        labelled, cysteine engineered antibody; and    -   (ii) measuring the binding of the affinity labelled, cysteine        engineered antibody to a capture media.

Another aspect of the invention is a method of screening cysteineengineered antibodies with highly reactive, unpaired cysteine aminoacids for thiol reactivity comprising:

-   -   (a) introducing one or more cysteine amino acids into a parent        antibody in order to generate a cysteine engineered antibody;    -   (b) reacting the cysteine engineered antibody with a        thiol-reactive affinity reagent to generate an affinity        labelled, cysteine engineered antibody; and    -   (c) measuring the binding of the affinity labelled, cysteine        engineered antibody to a capture media; and    -   (d) determining the thiol reactivity of the cysteine engineered        antibody with the thiol-reactive reagent.

Step (a) of the method of screening cysteine engineered antibodies maycomprise:

-   -   (i) mutagenizing a nucleic acid sequence encoding the cysteine        engineered antibody;    -   (ii) expressing the cysteine engineered antibody; and    -   (iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of screening cysteine engineered antibodies maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of screening cysteine engineered antibodies mayalso comprise:

-   -   (i) reacting the cysteine engineered antibody with a        thiol-reactive affinity reagent to generate an affinity        labelled, cysteine engineered antibody; and    -   (ii) measuring the binding of the affinity labelled, cysteine        engineered antibody to a capture media.

Cysteine engineered antibodies may be useful in the treatment of cancerand include antibodies specific for cell surface and transmembranereceptors, and tumor-associated antigens (TAA). Such antibodies may beused as naked antibodies (unconjugated to a drug or label moiety) or asFormula I antibody-drug conjugates (ADC).

Embodiments of the methods for preparing and screening a cysteineengineered antibody include where the parent antibody is an antibodyfragment, such as hu4D5Fabv8. The parent antibody may also be a fusionprotein comprising an albumin-binding peptide sequence (ABP). The parentantibody may also be a humanized antibody selected from huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7and huMAb4D5-8 (trastuzumab).

Cysteine engineered antibodies of the invention may be site-specificallyand efficiently coupled with a thiol-reactive reagent. Thethiol-reactive reagent may be a multifunctional linker reagent, acapture label reagent, a fluorophore reagent, or a drug-linkerintermediate.

The cysteine engineered antibody may be labeled with a detectable label,immobilized on a solid phase support and/or conjugated with a drugmoiety.

Another aspect of the invention is an antibody-drug conjugate compoundcomprising a cysteine engineered antibody (Ab), and a drug moiety (D)wherein the cysteine engineered antibody is attached through one or morefree cysteine amino acids by a linker moiety (L) to D; the compoundhaving Formula I:

Ab-(L-D)_(p)  I

where p is 1, 2, 3, or 4; and wherein the cysteine engineered antibodyis prepared by a process comprising replacing one or more amino acidresidues of a parent antibody by one or more free cysteine amino acids.Drug moieties include, but are not limited to a maytansinoid, anauristatin, a dolastatin, a trichothecene, CC1065, a calicheamicin andother enediyne antibiotics, a taxane, an anthracycline, andstereoisomers, isosteres, analogs or derivatives thereof. Exemplary drugmoieties include DM1, MMAE, and MMAF.

The antibody-drug conjugate of Formula I may further comprise analbumin-binding peptide (ABP) sequence; the composition having FormulaIa:

ABP-Ab-(L-D)_(p)  Ia

Another aspect of the invention is a composition comprising a cysteineengineered antibody or a cysteine engineered antibody-drug conjugate anda physiologically or pharmaceutically acceptable carrier or diluent.This composition for therapeutic use is sterile and may be lyophilized.

Another aspect of the invention includes diagnostic and therapeutic usesfor the compounds and compositions disclosed herein. Pharmaceuticalcompositions include combinations of Formula I compounds and one or morechemotherapeutic agents.

Another aspect of the invention is a method for killing or inhibitingthe proliferation of tumor cells or cancer cells comprising treating thecells with an amount of an antibody-drug conjugate of the invention, ora pharmaceutically acceptable salt or solvate thereof, being effectiveto kill or inhibit the proliferation of the tumor cells or cancer cells.

Other aspects of the invention include methods for treating: cancer; anautoimmune disease; or an infectious disease comprising administering toa patient in need thereof an effective amount of the antibody-drugconjugate compound of the invention, or a pharmaceutically acceptablesalt or solvate thereof.

Another aspect of the invention is a method for the treatment of cancerin a mammal, wherein the cancer is characterized by the overexpressionof an ErbB receptor. The mammal optionally does not respond, or respondspoorly, to treatment with an unconjugated anti-ErbB antibody. The methodcomprises administering to the mammal a therapeutically effective amountof an antibody-drug conjugate compound of the invention.

Another aspect of the invention is a method of inhibiting the growth oftumor cells that overexpress a growth factor receptor selected from thegroup consisting of HER2 receptor and EGF receptor comprisingadministering to a patient an antibody-drug conjugate compound whichbinds specifically to said growth factor receptor and a chemotherapeuticagent wherein said antibody-drug conjugate and said chemotherapeuticagent are each administered in amounts effective to inhibit growth oftumor cells in the patient.

Another aspect of the invention is a method for the treatment of a humanpatient susceptible to or diagnosed with a disorder characterized byoverexpression of ErbB2 receptor, comprising administering an effectiveamount of a combination of an antibody-drug conjugate compound and achemotherapeutic agent.

Another aspect of the invention is an assay method for detecting cancercells comprising: exposing cells to an antibody-drug conjugate compound,and determining the extent of binding of the antibody-drug conjugatecompound to the cells.

Another aspect of the invention is an article of manufacture comprisingan antibody-drug conjugate compound; a container; and a package insertor label indicating that the compound can be used to treat cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a three-dimensional representation of the hu4D5Fabv7antibody fragment derived by X-ray crystal coordinates. The structurepositions of the exemplary engineered Cys residues of the heavy andlight chains are numbered (according to a sequential numbering system).

FIG. 1B shows a sequential numbering scheme (top row), starting at theN-terminus in comparison with the Kabat numbering scheme (bottom row)for 4D5v7fabH. Kabat numbering insertions are noted by a,b,c.

FIGS. 2A and 2B show binding measurements with detection of absorbanceat 450 nm of hu4D5Fabv8 and hu4D5Fabv8 Cys mutant (ThioFab) phagevariants: (A) non-biotinylated phage-hu4D5Fabv8 and (B) biotinylatedphage-hu4D5Fabv8 (B) by the PHESELECTOR assay for interactions with BSA(open bar), HER2 (striped bar) or streptavidin (solid bar).

FIGS. 3A and 3B show binding measurements with detection of absorbanceat 450 nm of hu4D5Fabv8 (left) and hu4D5Fabv8 Cys mutant (ThioFab)variants: (A) non-biotinylated phage-hu4D5Fabv8 and (B) biotinylatedphage-hu4D5Fabv8 by the PHESELECTOR assay for interactions with: BSA(open bar), HER2 (striped bar) and streptavidin (solid bar). Light chainvariants are on the left side and heavy chain variants are on the rightside. Thiol reactivity=OD_(450 nm) for streptavidin binding÷OD_(450 nm)for HER2 (antibody) binding

FIG. 4A shows Fractional Surface Accessibility values of residues onwild type hu4D5Fabv8. Light chain sites are on the left side and heavychain sites are on the right side.

FIG. 4B shows binding measurements with detection of absorbance at 450nm of biotinylated hu4D5Fabv8 (left) and hu4D5Fabv8 Cys mutant (ThioFab)variants for interactions with HER2 (day 2), streptavidin (SA) (day 2),HER2 (day 4), and SA (day 4). Phage-hu4D5Fabv8 Cys variants wereisolated and stored at 4° C. Biotin conjugation was carried out eitherat day 2 or day 4 followed by PHESELECTOR analyses to monitor theirinteraction with Her2 and streptavidin as described in Example 2, andprobe the stability of reactive thiol groups on engineered ThioFabvariants.

FIG. 5 shows binding measurements with detection of absorbance at 450 nmof biotin-maleimide conjugated-hu4D5Fabv8 (A121C) and non-biotinylatedwild type hu4D5Fabv8 for binding to streptavidin and HER2. Each Fab wastested at 2 ng and 20 ng.

FIG. 6 shows ELISA analysis with detection of absorbance at 450 nm ofbiotinylated ABP-hu4D5Fabv8 wild type (wt), and ABP-hu4D5Fabv8 cysteinemutants V110C and A121C for binding with rabbit albumin, streptavidin(SA), and HER2.

FIG. 7 shows ELISA analysis with detection of absorbance at 450 nm ofbiotinylated ABP-hu4D5Fabv8 cysteine mutants (ThioFab variants): (leftto right) single Cys variants ABP-V110C, ABP-A121C, and double Cysvariants ABP-V110C-A88C and ABP-V110C-A121C for binding with rabbitalbumin, HER2 and streptavidin (SA), and probing with Fab-HRP or SA-HRP.

FIG. 8 shows binding of biotinylated ThioFab phage and an anti-phage HRPantibody to HER2 (top) and Streptavidin (bottom).

FIG. 9 shows an exemplary representation of an ABP-ThioFab fusionprotein drug conjugate binding to a HER2 receptor antigen. ABP=albuminbinding protein.

FIG. 10 shows an in vitro, cell proliferation assay of SK-BR-3 cellstreated with --trastuzumab; -▴- trastuzumab-SMCC-DM1; and -♦-hu4D5Fabv8 cysteine mutant-(A121C)-BMPEO-DM1.

FIG. 11 shows an in vitro, cell proliferation assay of SK-BR-3 cellstreated with: -∘-trastuzumab; -- trastuzumab-SMCC-DM1; and -□-hu4D5Fabv8 cysteine mutant (V110C)-BMPEO-DM1.

FIG. 12 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2Fo5 mammary tumor allografts, dosed on Day 0 with:

Vehicle (Buffer); -▪- ABP-hu4D5Fabv8 cysteine mutant (V110C lightchain)-DM1; and -- ABP-hu4D5Fabv8 cysteine mutant (A121C heavychain)-DM1.

FIG. 13A shows a cartoon depiction of biotinylated antibody binding toimmobilized HER2 with binding of HRP labeled secondary antibody forabsorbance detection.

FIG. 13B shows binding measurements with detection of absorbance at 450nm of biotin-maleimide conjugated thio-trastuzumab variants andnon-biotinylated wild type trastuzumab in binding to immobilized HER2.From left to right: V110C (single cys), A121C (single cys), V110C/A121C(double cys), and trastuzumab. Each thio IgG variant and trastuzumab wastested at 1, 10, and 100 ng.

FIG. 14A shows a cartoon depiction of biotinylated antibody binding toimmobilized HER2 with binding of biotin to anti-IgG-HRP for absorbancedetection.

FIG. 14B shows binding measurements with detection of absorbance at 450nm of biotin-maleimide conjugated-thio trastuzumab variants andnon-biotinylated wild type trastuzumab in binding to immobilizedstreptavidin. From left to right: V110C (single cys), A121C (singlecys), V110C/A121C (double cys), and trastuzumab. Each thio IgG variantand trastuzumab was tested at 1, 10, and 100 ng.

FIG. 15 shows the general process to prepare a cysteine engineeredantibody (ThioMab) expressed from cell culture for conjugation.

FIG. 16 shows non-reducing (top) and reducing (bottom) denaturingpolyacrylamide gel electrophoresis analysis of 2H9 ThioMab Fc variants(left to right, lanes 1-9): A339C; S337C; S324C; A287C; V284C; V282C;V279C; V273C, and 2H9 wild type after purification on immobilizedProtein A. The lane on the right is a size marker ladder, indicating theintact proteins are about 150 kDa, heavy chain fragments about 50 kDa,and light chain fragments about 25 kDa.

FIG. 17A shows non-reducing (left) and reducing (+DTT) (right)denaturing polyacrylamide gel electrophoresis analysis of 2H9 ThioMabvariants (left to right, lanes 1-4): L-V15C; S179C; S375C; S400C, afterpurification on immobilized Protein A.

FIG. 17B shows non-reducing (left) and reducing (+DTT) (right)denaturing polyacrylamide gel electrophoresis analysis of 2H9 and 3A5ThioMab variants after purification on immobilized Protein A.

FIG. 18 shows western blot analysis of biotinylated Thio-IgG variants.2H9 and 3A5 ThioMab variants were analyzed on reduced denaturingpolyacrylamide gel electrophoresis, the proteins were transferred tonitrocellulose membrane. The presence of antibody and conjugated biotinwere probed with anti-IgG-HRP (top) and streptavidin-HRP (bottom),respectively. Lane 1: 3A5H-A121C. Lane 2: 3A5 L-V110C. Lane 3:2H9H-A121C. Lane 4: 2H9L-V110C. Lane 5: 2H9 wild type.

FIG. 19 shows ELISA analysis for the binding of biotinylated 2H9variants to streptavidin by probing with anti-IgG-HRP and measuring theabsorbance at 450 nm of (top bar diagram). Bottom schematic diagramdepicts the experimental design used in the ELISA analysis.

FIG. 20 shows an in vitro, cell proliferation assay of SK-BR-3 cellstreated with: --trastuzumab; -▴- trastuzumab-SMCC-DM1 with a drugloading of 3.4 DM1/Ab; and -♦- thio-trastuzumab (A121C)-BMPEO-DM1 with adrug loading of 1.6 DM1/Ab.

FIG. 21A shows an in vitro, cell proliferation assay of HT 1080EphB2cells treated with:

parent 2H9 anti-EphB2R; and -□- thio 2H9 (A121C) BMPEO-DM1.

FIG. 21B shows an in vitro, cell proliferation assay of BT 474 cellstreated with:

parent 2H9 anti-EphB2R; and -□- thio 2H9 (A121C) BMPEO-DM1.

FIG. 22 shows an in vitro, cell proliferation assay of PC3/neo cellstreated with: -♦-3A5 anti MUC16-SMCC-DM1; and -▪- thio 3A5 (A121C)BMPEO-DM1.

FIG. 23 shows an in vitro, cell proliferation assay of PC3/MUC16 cellstreated with: -♦-3A5 anti MUC16-SMCC-DM1; and -▪- thio 3A5 (A121C)BMPEO-DM1.

FIG. 24 shows an in vitro, cell proliferation assay of OVCAR-3 cellstreated with: -♦-3A5 anti MUC16-SMCC-DM1; and -▪- thio 3A5 (A121C)BMPEO-DM1.

FIG. 25 shows the mean tumor volume change over 21 days in athymic nudemice with MMTV-HER2Fo5 mammary tumor allografts, after a single dose onDay 0 with:

Vehicle (Buffer); -- trastuzumab-SMCC-DM1 10 mg/kg, with a drug loadingof 3.4 DM1/Ab; -▪- thio trastuzumab (A121C)—SMCC-DM1 21 mg/kg, with adrug loading of 1.6 DM1/Ab; and -□- thio trastuzumab (A121C)—SMCC-DM110mg/kg, with a drug loading of 1.6 DM1/Ab.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, and are consistent with:Singleton et al (1994) Dictionary of Microbiology and Molecular Biology,2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; minibodies (Olafsen et al (2004) ProteinEng. Design & Sel. 17(4):315-323), fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anyof the above which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. No. 4,816,567; U.S.Pat. No. 5,807,715). The monoclonal antibodies may also be isolated fromphage antibody libraries using the techniques described in Clackson etal (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al (1998) J. Immunol.161:4083-4090; Lund et al (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

An “ErbB receptor” is a receptor protein tyrosine kinase which belongsto the ErbB receptor family whose members are important mediators ofcell growth, differentiation and survival. The ErbB receptor familyincludes four distinct members including epidermal growth factorreceptor (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) andHER4 (ErbB4 or tyro2). A panel of anti-ErbB2 antibodies has beencharacterized using the human breast tumor cell line SKBR3 (Hudziak etal (1989) Mol. Cell. Biol. 9(3):1165-1172. Maximum inhibition wasobtained with the antibody called 4D5 which inhibited cellularproliferation by 56%. Other antibodies in the panel reduced cellularproliferation to a lesser extent in this assay. The antibody 4D5 wasfurther found to sensitize ErbB2-overexpressing breast tumor cell linesto the cytotoxic effects of TNF-a (U.S. Pat. No. 5,677,171). Theanti-ErbB2 antibodies discussed in Hudziak et al. are furthercharacterized in Fendly et al (1990) Cancer Research 50:1550-1558; Kottset al. (1990) In Vitro 26(3):59A; Sarup et al. (1991) Growth Regulation1:72-82; Shepard et al. J. (1991) Clin. Immunol. 11 (3):117-127; Kumaret al. (1991) Mol. Cell. Biol. 11(2):979-986; Lewis et al. (1993) CancerImmunol. Immunother. 37:255-263; Pietras et al. (1994) Oncogene9:1829-1838; Vitetta et al. (1994) Cancer Research 54:5301-5309;Sliwkowski et al. (1994) J. Biol. Chem. 269(20):14661-14665; Scott etal. (1991) J. Biol. Chem. 266:14300-5; D'souza et al. Proc. Natl. Acad.Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research56:1457-1465; and Schaefer et al. (1997) Oncogene 15:1385-1394.

The ErbB receptor will generally comprise an extracellular domain, whichmay bind an ErbB ligand; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.The ErbB receptor may be a “native sequence” ErbB receptor or an “aminoacid sequence variant” thereof. Preferably, the ErbB receptor is nativesequence human ErbB receptor. Accordingly, a “member of the ErbBreceptor family” is EGFR (ErbB1), ErbB2, ErbB3, ErbB4 or any other ErbBreceptor currently known or to be identified in the future.

The terms “ErbB1”, “epidermal growth factor receptor”, “EGFR” and “HER1”are used interchangeably herein and refer to EGFR as disclosed, forexample, in Carpenter et al (1987) Ann. Rev. Biochem., 56:881-914,including naturally occurring mutant forms thereof (e.g., a deletionmutant EGFR as in Humphrey et al (1990) Proc. Nat. Acad. Sci. (USA)87:4207-4211). The term erbB1 refers to the gene encoding the EGFRprotein product. Antibodies against HER1 are described, for example, inMurthy et al (1987) Arch. Biochem. Biophys., 252:549-560 and in WO95/25167.

The term “ERRP”, “EGF-Receptor Related Protein”, “EGFR Related Protein”and “epidermal growth factor receptor related protein” are usedinterchangeably herein and refer to ERRP as disclosed, for example inU.S. Pat. No. 6,399,743 and US Publication No. 2003/0096373.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al(1985) Proc. Nat. Acad. Sci. (USA) 82:6497-6501 and Yamamoto et al(1986) Nature, 319:230-234 (Genebank accession number X03363). The term“erbB2” refers to the gene encoding human ErbB2 and “neu” refers to thegene encoding rat p185neu. Preferred ErbB2 is native sequence humanErbB2.

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal (1989) Proc. Nat. Acad. Sci. (USA) 86:9193-9197. Antibodies againstErbB3 are known in the art and are described, for example, in U.S. Pat.Nos. 5,183,884, 5,480,968 and in WO 97/35885.

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Application No 599,274; Plowman et al(1993) Proc. Natl. Acad. Sci. USA 90:1746-1750; and Plowman et al (1993)Nature 366:473-475, including isoforms thereof, e.g., as disclosed in WO99/19488. Antibodies against HER4 are described, for example, in WO02/18444.

Antibodies to ErbB receptors are available commercially from a number ofsources, including, for example, Santa Cruz Biotechnology, Inc.,California, USA.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% sequence identity with at least one receptor bindingdomain of a native ErbB ligand or with at least one ligand bindingdomain of a native ErbB receptor, and preferably, they will be at leastabout 80%, more preferably, at least about 90% homologous by sequencewith such receptor or ligand binding domains. The amino acid sequencevariants possess substitutions, deletions, and/or insertions at certainpositions within the amino acid sequence of the native amino acidsequence. Amino acids are designated by the conventional names,one-letter and three-letter codes.

“Sequence identity” is defined as the percentage of residues in theamino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Methods and computer programs for thealignment are well known in the art. One such computer program is “Align2,” authored by Genentech, Inc., which was filed with user documentationin the United States Copyright Office, Washington, D.C. 20559, on Dec.10, 1991.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, (1991) “Annu. Rev. Immunol.” 9:457-92.To assess ADCC activity of a molecule of interest, an in vitro ADCCassay, such as that described in U.S. Pat. No. 5,500,362 and U.S. Pat.No. 5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al (1998) PROC. NAT. ACAD. SCl.(USA) (USA) 95:652-656.

“Human effector cells” are leukocytes which express one or more constantregion receptors (FcRs) and perform effector functions. Preferably, thecells express at least FcγRIII and perform ADCC effector function.Examples of human leukocytes which mediate ADCC include peripheral bloodmononuclear cells (PBMC), natural killer (NK) cells, monocytes,cytotoxic T cells and neutrophils; with PBMCs and NK cells beingpreferred. The effector cells may be isolated from a native sourcethereof, e.g., from blood or PBMCs as described herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc constant region of an antibody. The preferred FcR is anative sequence human FcR. Moreover, a preferred FcR is one which bindsan IgG antibody (a gamma receptor) and includes receptors of the FcγRI,FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See review M. in Da{tilde over (e)}ron, “Annu. Rev.Immunol.” 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet,“Annu. Rev. Immunol”., 9:457-92 (1991); Capel et al (1994) Immunomethods4:25-34; and de Haas et al (1995) J. Lab. Clin. Med. 126:330-41. OtherFcRs, including those to be identified in the future, are encompassed bythe term “FcR” herein. The term also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al (1976) J. Immunol., 117:587 and Kim et al (1994) J.Immunol. 24:249).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g., an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g., as described in Gazzano-Santoro et al J. Immunol. Methods, 202:163(1996), may be performed.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al supra) and/or those residues from a “hypervariableloop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol.,196:901-917). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). Anti-ErbB2 antibody scFv fragments are described in WO 93/16185;U.S. Pat. Nos. 5,571,894; and 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(VH) connected to a variable light domain (VL) in the same polypeptidechain (VH−VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al (1993) Proc. Natl.Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Humanization is a method to transfer the murine antigenbinding information to a non-immunogenic human antibody acceptor, andhas resulted in many therapeutically useful drugs. The method ofhumanization generally begins by transferring all six murinecomplementarity determining regions (CDRs) onto a human antibodyframework (Jones et al, (1986) Nature 321:522-525). These CDR-graftedantibodies generally do not retain their original affinity for antigenbinding, and in fact, affinity is often severely impaired. Besides theCDRs, select non-human antibody framework residues must also beincorporated to maintain proper CDR conformation (Chothia et al (1989)Nature 342:877). The transfer of key mouse framework residues to thehuman acceptor in order to support the structural conformation of thegrafted CDRs has been shown to restore antigen binding and affinity(Riechmann et al (1992) J. Mol. Biol. 224, 487-499; Foote and Winter,(1992) J. Mol. Biol. 224:487-499; Presta et al (1993) J. Immunol. 151,2623-2632; Werther et al (1996) J. Immunol. Methods 157:4986-4995; andPresta et al (2001) Thromb. Haemost. 85:379-389). For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a hypervariable region of the recipient are replacedby residues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances,framework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see U.S. Pat. No. 6,407,213; Joneset al (1986) Nature, 321:522-525; Riechmann et al (1988) Nature332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as an intramolecular or intermoleculardisulfide bridge.

The term “thiol reactivity value” is a quantitative characterization ofthe reactivity of free cysteine amino acids. The thiol reactivity valueis the percentage of a free cysteine amino acid in a cysteine engineeredantibody which reacts with a thiol-reactive reagent, and converted to amaximum value of 1. For example, a free cysteine amino acid on acysteine engineered antibody which reacts in 100% yield with athiol-reactive reagent, such as a biotin-maleimide reagent, to form abiotin-labelled antibody has a thiol reactivity value of 1.0. Anothercysteine amino acid engineered into the same or different parentantibody which reacts in 80% yield with a thiol-reactive reagent has athiol reactivity value of 0.8. Another cysteine amino acid engineeredinto the same or different parent antibody which fails totally to reactwith a thiol-reactive reagent has a thiol reactivity value of 0.Determination of the thiol reactivity value of a particular cysteine maybe conducted by ELISA assay, mass spectroscopy, liquid chromatography,autoradiography, or other quantitative analytical tests.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody may be directed against a target antigenof interest, e.g. a biologically important polypeptide. Antibodiesdirected against nonpolypeptide antigens (such as tumor-associatedglycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated.

Exemplary parent antibodies include antibodies having affinity andselectivity for cell surface and transmembrane receptors andtumor-associated antigens (TAA).

Other exemplary parent antibodies include those selected from, andwithout limitation, anti-estrogen receptor antibody, anti-progesteronereceptor antibody, anti-p53 antibody, anti-HER-2/neu antibody, anti-EGFRantibody, anti-cathepsin D antibody, anti-Bcl-2 antibody,anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody,anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoproteinantibody, anti-CEA antibody, anti-retinoblastoma protein antibody,anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody,anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13 antibody,anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody,anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35antibody, anti-CD38 antibody, anti-CD41 antibody, anti-LCA/CD45antibody, anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39antibody, anti-CD100 antibody, anti-CD95/Fas antibody, anti-CD99antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71antibody, anti-c-myc antibody, anti-cytokeratins antibody,anti-vimentins antibody, anti-HPV proteins antibody, anti-kappa lightchains antibody, anti-lambda light chains antibody, anti-melanosomesantibody, anti-prostate specific antigen antibody, anti-S-100 antibody,anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibodyand anti-Tn-antigen antibody.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody “which binds” a molecular target or an antigen of interest,e.g., ErbB2 antigen, is one capable of binding that antigen withsufficient affinity such that the antibody is useful in targeting a cellexpressing the antigen. Where the antibody is one which binds ErbB2, itwill usually preferentially bind ErbB2 as opposed to other ErbBreceptors, and may be one which does not significantly cross-react withother proteins such as EGFR, ErbB3 or ErbB4. In such embodiments, theextent of binding of the antibody to these non-ErbB2 proteins (e.g.,cell surface binding to endogenous receptor) will be less than 10% asdetermined by fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). Sometimes, the anti-ErbB2 antibody willnot significantly cross-react with the rat neu protein, e.g., asdescribed in Schecter et al. (1984) Nature 312:513 and Drebin et al(1984) Nature 312:545-548.

Molecular targets for antibodies encompassed by the present inventioninclude CD proteins and their ligands, such as, but not limited to: (i)CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40, CD79α (CD79a), and CD79β(CD79b); (ii) members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; (iii) cell adhesion moleculessuch as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and αv/β3 integrin,including either alpha or beta subunits thereof (e.g. anti-CD11a,anti-CD18 or anti-CD11b antibodies); (iv) growth factors such as VEGF;IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor;mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7 etc; and(v) cell surface and transmembrane tumor-associated antigens (TAA).

Unless indicated otherwise, the term “monoclonal antibody 4D5” refers toan antibody that has antigen binding residues of, or derived from, themurine 4D5 antibody (ATCC CRL 10463). For example, the monoclonalantibody 4D5 may be murine monoclonal antibody 4D5 or a variant thereof,such as a humanized 4D5. Exemplary humanized 4D5 antibodies includehuMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,huMAb4D5-7 and huMAb4D5-8 (trastuzumab, HERCEPTIN®) as in U.S. Pat. No.5,821,337.

The terms “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as the development or spread of cancer. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small- cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

An “ErbB-expressing cancer” is one comprising cells which have ErbBprotein present at their cell surface. An “ErbB2-expressing cancer” isone which produces sufficient levels of ErbB2 at the surface of cellsthereof, such that an anti-ErbB2 antibody can bind thereto and have atherapeutic effect with respect to the cancer.

A cancer which “overexpresses” an antigenic receptor is one which hassignificantly higher levels of the receptor, such as ErbB2, at the cellsurface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. Receptor overexpression may bedetermined in a diagnostic or prognostic assay by evaluating increasedlevels of the receptor protein present on the surface of a cell (e.g.,via an immunohistochemistry assay; IHC). Alternatively, or additionally,one may measure levels of receptor-encoding nucleic acid in the cell,e.g., via fluorescent in situ hybridization (FISH; see WO 98/45479),southern blotting, or polymerase chain reaction (PCR) techniques, suchas real time quantitative PCR (RT-PCR).

The tumors overexpressing ErbB2 (HER2) are rated by immunohistochemicalscores corresponding to the number of copies of HER2 molecules expressedper cell, and can been determined biochemically: 0=0-10,000 copies/cell,1+=at least about 200,000 copies/cell, 2+=at least about 500,000copies/cell, 3+=about 1−2×10⁶ copies/cell. Overexpression of HER2 at the3+level, which leads to ligand-independent activation of the tyrosinekinase (Hudziak et al (1987) Proc. Natl. Acad. Sci. USA, 84:7159-7163),occurs in approximately 30% of breast cancers, and in these patients,relapse-free survival and overall survival are diminished (Slamon et al(1989) Science, 244:707-712; Slamon et al (1987) Science, 235:177-182).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, andradioactive isotopes of Lu), chemotherapeutic agents, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including synthetic analogs andderivatives thereof.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or organs or a co-segregateor manifestation thereof or resulting condition therefrom. In many ofthese autoimmune and inflammatory disorders, a number of clinical andlaboratory markers may exist, including, but not limited to,hypergammaglobulinemia, high levels of autoantibodies, antigen-antibodycomplex deposits in tissues, benefit from corticosteroid orimmunosuppressive treatments, and lymphoid cell aggregates in affectedtissues. Without being limited to any one theory regarding B-cellmediated autoimmune disease, it is believed that B cells demonstrate apathogenic effect in human autoimmune diseases through a multitude ofmechanistic pathways, including autoantibody production, immune complexformation, dendritic and T-cell activation, cytokine synthesis, directchemokine release, and providing a nidus for ectopic neo-lymphogenesis.Each of these pathways may participate to different degrees in thepathology of autoimmune diseases. An autoimmune disease can be anorgan-specific disease (i.e., the immune response is specificallydirected against an organ system such as the endocrine system, thehematopoietic system, the skin, the cardiopulmonary system, thegastrointestinal and liver systems, the renal system, the thyroid, theears, the neuromuscular system, the central nervous system, etc.) or asystemic disease which can affect multiple organ systems (for example,systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis,etc.).

The term “cytostatic” refers to the effect of limiting the function ofcells, such as limiting cellular growth or proliferation of cells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADEL™,Millenium Pharm.), Fulvestrant (FASLODEX®, Astrazeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline),Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), andGefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen),alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, including dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, anthramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKR polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY 117018, onapristone, and FARESTON.toremifene; (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA®letrozole, and ARIMIDEX® anastrozole; (iii) anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinaseinhibitors; (vii) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;(viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; (ix) vaccines such as genetherapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine,and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptablesalts, acids or derivatives of any of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBITUX®) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF (see WO 98/50433, Abgenix). The anti-EGFR antibodymay be conjugated with a cytotoxic agent, thus generating animmunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH). Examplesof small molecules that bind to EGFR include ZD 1839 or Gefitinib(IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™;Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).

Protein kinase inhibitors include tyrosine kinase inhibitors whichinhibits to some extent tyrosine kinase activity of a tyrosine kinasesuch as an ErbB receptor. Examples of such inhibitors include theEGFR-targeted drugs noted in the preceding paragraph as well asquinazolines such as PD 153035,4-(3-chloroanilino) quinazoline,pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g., those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG);pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (Gleevec; Novartis); PKI 166 (Novartis);GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib(Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11(Imclone); or as described in any of the following patent publications:WO 99/09016 (American Cyanamid); WO 98/43960 (American Cyanamid); WO97/38983 (Warner Lambert); WO 99/06378 (Warner Lambert); WO 99/06396(Warner Lambert); WO 96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO96/3397 (Zeneca); and WO 96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The preferred anti-angiogenic factorherein is an antibody that binds to Vascular Endothelial Growth Factor(VEGF).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically or hydrolytically activated or converted into themore active parent form. See, e.g., Wilman, “Prodrugs in CancerChemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615thMeeting Belfast (1986) and Stella et al “Prodrugs: A Chemical Approachto Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al(ed.), pp. 247-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the anti-ErbB2 antibodies disclosed herein and, optionally, achemotherapeutic agent) to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to a coat protein on the surface of phage,e.g., filamentous phage, particles. One utility of phage display lies inthe fact that large libraries of randomized protein variants can berapidly and efficiently sorted for those sequences that bind to a targetmolecule with high affinity. Display of peptide and protein libraries onphage has been used for screening millions of polypeptides for ones withspecific binding properties. Polyvalent phage display methods have beenused for displaying small random peptides and small proteins, typicallythrough fusions to either pIII or pVIII of filamentous phage (Wells andLowman, (1992) Curr. Opin. Struct. Biol., 3:355-362, and referencescited therein). In monovalent phage display, a protein or peptidelibrary is fused to a phage coat protein or a portion thereof, andexpressed at low levels in the presence of wild type protein. Avidityeffects are reduced relative to polyvalent phage so that sorting is onthe basis of intrinsic ligand affinity, and phagemid vectors are used,which simplify DNA manipulations. Lowman and Wells, Methods: A companionto Methods in Enzymology, 3:205-0216 (1991). Phage display includestechniques for producing antibody-like molecules (Janeway, C., Travers,P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York, p627-628; Lee et al).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments, a linker is specified as L.Linkers include a divalent radical such as an alkyldiyl, an arylene, aheteroarylene, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

The term “label” means any moiety which can be covalently attached to anantibody and that functions to: (i) provide a detectable signal; (ii)interact with a second label to modify the detectable signal provided bythe first or second label, e.g. FRET (fluorescence resonance energytransfer); (iii) stabilize interactions or increase affinity of binding,with antigen or ligand; (iv) affect mobility, e.g. electrophoreticmobility, or cell-permeability, by charge, hydrophobicity, shape, orother physical parameters, or (v) provide a capture moiety, to modulateligand affinity, antibody/antigen binding, or ionic complexation.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and 1 or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an ADC.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis -(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and an ADC. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

The following abbreviations are used herein and have the indicateddefinitions: BME is beta-mercaptoethanol, Boc is N-(t-butoxycarbonyl),cit is citrulline (2-amino-5-ureido pentanoic acid), dap is dolaproine,DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4-dimethylaminopyridine, DME isethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF isN,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid),DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl),nor is(1S,2R)-(+)-norephedrine, PAB is p-aminobenzylcarbamoyl, PBS isphosphate-buffered saline (pH 7), PEG is polyethylene glycol, Ph isphenyl, Pnp is p-nitrophenyl, MC is 6-maleimidocaproyl, phe isL-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphoniumhexafluorophosphate, SEC is size-exclusion chromatography, Su issuccinimide, TFA is trifluoroacetic acid, TLC is thin layerchromatography, UV is ultraviolet, and val is valine.

Cysteine Engineered Antibodies

The compounds of the invention include cysteine engineered antibodieswhere one or more amino acids of a wild-type or parent antibody arereplaced with a cysteine amino acid. Any form of antibody may be soengineered, i.e. mutated. For example, a parent Fab antibody fragmentmay be engineered to form a cysteine engineered Fab, referred to hereinas “ThioFab.” Similarly, a parent monoclonal antibody may be engineeredto form a “ThioMab.” It should be noted that a single site mutationyields a single engineered cysteine residue in a ThioFab, while a singlesite mutation yields two engineered cysteine residues in a ThioMab, dueto the dimeric nature of the IgG antibody. Mutants with replaced(“engineered”) cysteine (Cys) residues are evaluated for the reactivityof the newly introduced, engineered cysteine thiol groups. The thiolreactivity value is a relative, numerical term in the range of 0 to 1.0and can be measured for any cysteine engineered antibody. Thiolreactivity values of cysteine engineered antibodies of the invention arein the ranges of 0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0.

The design, selection, and preparation methods of the invention enablecysteine engineered antibodies which are reactive with electrophilicfunctionality. These methods further enable antibody conjugate compoundssuch as antibody-drug conjugate (ADC) compounds with drug molecules atdesignated, designed, selective sites. Reactive cysteine residues on anantibody surface allow specifically conjugating a drug moiety through athiol reactive group such as maleimide or haloacetyl. The nucleophilicreactivity of the thiol functionality of a Cys residue to a maleimidegroup is about 1000 times higher compared to any other amino acidfunctionality in a protein, such as amino group of lysine residues orthe N-terminal amino group. Thiol specific functionality in iodoacetyland maleimide reagents may react with amine groups, but higher pH (>9.0)and longer reaction times are required (Garman, 1997, Non-RadioactiveLabelling: A Practical Approach, Academic Press, London).

Cysteine engineered antibodies of the invention preferably retain theantigen binding capability of their wild type, parent antibodycounterparts. Thus, cysteine engineered antibodies are capable ofbinding, preferably specifically, to antigens. Such antigens include,for example, tumor-associated antigens (TAA), cell surface receptorproteins and other cell surface molecules, transmembrane proteins,signalling proteins, cell survival regulatory factors, cellproliferation regulatory factors, molecules associated with (for e.g.,known or suspected to contribute functionally to) tissue development ordifferentiation, lymphokines, cytokines, molecules involved in cellcycle regulation, molecules involved in vasculogenesis and moleculesassociated with (for e.g., known or suspected to contribute functionallyto) angiogenesis. The tumor-associated antigen may be a clusterdifferentiation factor (i.e., a CD protein). An antigen to which acysteine engineered antibody is capable of binding may be a member of asubset of one of the above-mentioned categories, wherein the othersubset(s) of said category comprise other molecules/antigens that have adistinct characteristic (with respect to the antigen of interest).

The parent antibody may also be a humanized antibody selected fromhuMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6,huMAb4D5-7 and huMAb4D5-8 (Trastuzumab, HERCEPTIN®) as described inTable 3 of U.S. Pat. No. 5,821,337, expressly incorporated herein byreference; humanized 520C9 (WO 93/21319) and humanized 2C4 antibodies asdescribed herein.

Cysteine engineered antibodies of the invention may be site-specificallyand efficiently coupled with a thiol-reactive reagent. Thethiol-reactive reagent may be a multifunctional linker reagent, acapture, i.e. affinity, label reagent (e.g. a biotin-linker reagent), adetection label (e.g. a fluorophore reagent), a solid phaseimmobilization reagent (e.g. SEPHAROSE™, polystyrene, or glass), or adrug-linker intermediate. One example of a thiol-reactive reagent isN-ethyl maleimide (NEM). In an exemplary embodiment, reaction of aThioFab with a biotin-linker reagent provides a biotinylated ThioFab bywhich the presence and reactivity of the engineered cysteine residue maybe detected and measured. Reaction of a ThioFab with a multifunctionallinker reagent provides a ThioFab with a functionalized linker which maybe further reacted with a drug moiety reagent or other label. Reactionof a ThioFab with a drug-linker intermediate provides a ThioFab drugconjugate.

The exemplary methods described here may be applied generally to theidentification and production of antibodies, and more generally, toother proteins through application of the design and screening stepsdescribed herein.

Such an approach may be applied to the conjugation of otherthiol-reactive agents in which the reactive group is, for example, amaleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive conjugation partner (Haugland, 2003, Molecular ProbesHandbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in BioconjugateTechniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). Thepartner may be a cytotoxic agent (e.g. a toxin such as doxorubicin orpertussis toxin), a fluorophore such as a fluorescent dye likefluorescein or rhodamine, a chelating agent for an imaging orradiotherapeutic metal, a peptidyl or non-peptidyl label or detectiontag, or a clearance-modifying agent such as various isomers ofpolyethylene glycol, a peptide that binds to a third component, oranother carbohydrate or lipophilic agent.

The sites identified on the exemplary antibody fragment, hu4D5Fabv8,herein are primarily in the constant domain of an antibody which is wellconserved across all species of antibodies. These sites should bebroadly applicable to other antibodies, without further need ofstructural design or knowledge of specific antibody structures, andwithout interference in the antigen binding properties inherent to thevariable domains of the antibody.

Cysteine engineered antibodies which may be useful in the treatment ofcancer include, but are not limited to, antibodies against cell surfacereceptors and tumor-associated antigens (TAA). Such antibodies may beused as naked antibodies (unconjugated to a drug or label moiety) or asFormula I antibody-drug conjugates (ADC). Tumor-associated antigens areknown in the art, and can prepared for use in generating antibodiesusing methods and information which are well known in the art. Inattempts to discover effective cellular targets for cancer diagnosis andtherapy, researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of TAA include, but are not limited to, TAA (1)-(36) listedbelow. For convenience, information relating to these antigens, all ofwhich are known in the art, is listed below and includes names,alternative names, Genbank accession numbers and primary reference(s),following nucleic acid and protein sequence identification conventionsof the National Center for Biotechnology Information (NCBI). Nucleicacid and protein sequences corresponding to TAA (1)-(36) are availablein public databases such as GenBank. Tumor-associated antigens targetedby antibodies include all amino acid sequence variants and isoformspossessing at least about 70%, 80%, 85%, 90%, or 95% sequence identityrelative to the sequences identified in the cited references, or whichexhibit substantially the same biological properties or characteristicsas a TAA having a sequence found in the cited references. For example, aTAA having a variant sequence generally is able to bind specifically toan antibody that binds specifically to the TAA with the correspondingsequence listed. The sequences and disclosure in the referencespecifically recited herein are expressly incorporated by reference.

Tumor-associated Antigens (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_(—)001203)ten Dijke, P., et al Science 264 (5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004063362 (claim 2); WO2003042661 (claim 12);US2003134790-A1 (Page 38-39); WO2002102235 (claim 13; Page 296);WO2003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530);WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358(claim 1; Page 183); WO200254940 (Page 100-101); WO200259377(Page349-350); WO200230268 (claim 27; Page 376); WO200148204 (Example; FIG.4) NP_(—)001194 bone morphogenetic protein receptor, typeIB/pid=NP_(—)001194.1-Cross-references: MIM:603248; NP_(—)001194.1;AY065994(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486)Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395(6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (Example2); WO200299074 (claim 19; Page 127-129); WO200286443 (claim 27; Pages222, 393); WO2003003906 (claim 10; Page 293); WO200264798 (claim 33;Page 93-95); WO200014228 (claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (claim 12; Page 150); NP_(—)003477 solute carrier family 7(cationic amino acid transporter, y+ system), member5/pid=NP_(—)003477.3—Homo sapiens

Cross-references: MIM:600182; NP_(—)003477.3; NM_(—)015923;NM_(—)003486_(—)1

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_(—)012449)Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc.Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004065577 (claim 6);WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (claim 2);WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830 (Example5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619);WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG.2A);NP_(—)036581 six transmembrane epithelial antigen of the prostate

Cross-references: MIM:604415; NP_(—)036581.1; NM_(—)012449_(—)1

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486)J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14);WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page 116-121);US2003124140 (Example 16); Cross-references: GI:34501467; AAK74120.3;AF361486_(—)1(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_(—)005823) Yamaguchi, N., et al Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (claim 14);(WO2002102235 (claim 13; Page 287-288); WO2002101075 (claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_(—)005814.2; NM_(—)005823_(—)1(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter3b,Genbank accession no. NM_(—)006424)J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11);WO2002102235 (claim 13; Page 326); EP875569 (claim 1; Page 17-19);WO200157188 (claim 20; Page 329); WO2004032842 (Example IV); WO200175177(claim 24; Page 139-140);

Cross-references: MIM:604217; NP_(—)006415.1; NM_(—)006424_(—)1

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878)Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1);WO2003003984 (claim 1); WO200206339 (claim 1; Page 50); WO200188133(claim 1; Page 41-43, 48-58); WO2003054152 (claim 20); WO2003101400(claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew;HGNC:10737;(8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002)Cancer Res. 62:2546-2553; US2003129192 (claim 2); US2004044180 (claim12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056(Example 5); WO2003105758 (claim 12); US2003206918 (Example 5);EP1347046 (claim 1); WO2003025148 (claim 20);

Cross-references: GI:37182378; AAQ88991.1; AY358628_(—)1

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999;Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et alEur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79,1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995;Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et alHum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet.12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002)Hum. Genet. 111, 198-206; WO2004045516 (claim 1); WO2004048938 (Example2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475(claim 1); WO2003016475 (claim 1); WO200261087 (FIG. 1); WO2003016494(FIG. 6); WO2003025138 (claim 12; Page 144); WO200198351 (claim 1; Page124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; Page297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.5,773,223 (Claim 1a; Col 31-34); WO2004001004;(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_(—)017763);WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim12); WO2003083074 (claim 14; Page 61); WO2003018621 (claim 1);WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6);

Cross-references: LocusID:54894; NP_(—)060233.2; NM_(—)017763_(—)1

(11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,prostate cancer associated gene 1, prostate cancer associated protein 1,six transmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138)Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397(claim 1; FIG. 1); WO200272596 (claim 13; Page 54-55); WO200172962(claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16);US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12;FIG. 10);

Cross-references: GI:22655488; AAN04080.1; AF455138_(—)1

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_(—)017636)Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003143557 (claim 4); WO200040614 (claim 14; Page 100-103);WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268(claim 27; Page 391); US2003219806 (claim 4); WO200162794 (claim 14;FIG. 1A-D);

Cross-references: MIM:606936; NP_(—)060106.2; NM_(—)017636_(—)1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_(—)003203 or NM_(—)003212)Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (claim 1); WO2003083041(Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; Page 52-53);WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; Page 94-95, 105);WO200222808 (claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2);

Cross-references: MIM:187395; NP_(—)003203.1; NM_(—)003212_(—)1

(14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004)Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad.Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (claim9); WO2004045520 (Example 4); WO9102536 (FIG. 9.1-9.9); WO2004020595(claim 1);

Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79p, 1 Gb (immunoglobulin-associated beta), B29,Genbank accession no. NM_(—)000626 or 11038674)Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100(9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625);WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1,page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573(claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003048202 (claim 1,pages 306 and 309); WO 99/558658, U.S. Pat. No. 6,534,482 (claim 13,FIG. 17A/B); WO200055351 (claim 11, pages 1145-1146);

Cross-references: MIM:147245; NP_(—)000617.1; NM_(—)000626_(—)1

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP 1B, SPAPIC, Genbank accession no. NM_(—)030764,AY358130) Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (claim 2);WO2003077836; WO200138490 (claim 5; FIG. 18D-1-18D-2); WO2003097803(claim 12); WO2003089624 (claim 25);

Cross-references: MIM:606509; NP_(—)110391.2; NM_(—)030764_(—)1

(17) HER2 (ErbB2, Genbank accession no. M11730)Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., etal Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci.U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165,869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999;Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993)Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 11);WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIG.1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (claim 52; FIG. 7); WO200020579(claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (claim 3; Col 31-38);WO9630514 (claim 2; Page 56-61); EP1439393 (claim 7); WO2004043361(claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4);

Accession: P04626; EMBL; M11767; AAA35808.I. EMBL; M11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession no. M18728);Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc.Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393(claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim12); WO200278524 (Example 2); WO200286443 (claim 27; Page 427);WO200260317 (claim 2);

Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;

(19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl. Acad. Sci.U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (claim 1); WO200264798(claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);

Cross-references: MIM:179780; AAH 17023.1; BC017023_(—)1

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et alNature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001;Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003)Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (claim 1; Page 55-59);

Accession: □9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al GenomeRes. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci.U.S.A. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373(claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG.52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52);US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1;FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634(claim 1);(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_(—)004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (claim 12);WO200053216 (claim 1; Page 41); WO2004065576 (claim 1); WO2004020583(claim 9); WO2003004529 (Page 128-132); WO200053216 (claim 1; Page 42);

Cross-references: MIM:600997; NP_(—)004433.2; NM_(—)004442_(—)1

(23) ASLG659 (B7h, Genbank accession no. AX092328)US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3);US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (FIG.60); WO2002102235 (claim 13; Page 299); US2003091580 (Example 2);WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7b);WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54; Page 45-46);WO200206317 (Example 2; Page 320-321, claim 34; Page 321-322);WO200271928 (Page 468-469); WO200202587 (Example 1; FIG. 1); WO200140269(Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207);WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (Page233-234, 452-453); WO 0116318;(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436) Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (claim 17); WO2003008537 (claim 1);WO200281646 (claim 1; Page 164); WO2003003906 (claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; Page 97); WO9851824(claim 10; Page 94); WO9840403 (claim 2; FIG. 1B);

Accession: 043653; EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763);AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1—Homosapiens Species: Homo sapiens (human)WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example3, claim 20); US2003194704 (claim 45);

Cross-references: GI:30102449; AAP14954.1; AY260763_(—)1

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_(—)443177.1—Homosapiens Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (claim 3; Page 133); WO200224909(Example 3; FIG. 3);

Cross-references: MIM:606269; NP_(—)443177.1; NM_(—)052945_(—)1;AF132600

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991)J. Exp. Med. 173:137-146; WO2003072036 (claim 1; FIG. 1);

Cross-references: MIM:107266; NP_(—)001762.1; NM_(—)001771_(—)1

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pI: 4.84, MW: 25028 ™: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_(—)001774.10)WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S.Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531;Mueller et al (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al(1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al (1988) EMBO J. 7(11):3457-3464;(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pI: 8.54 MW: 41959 ™: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_(—)001707.1) WO2004040000; WO2004015426; US2003105292 (Example2); U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1);WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129(Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (claim1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931(pages 56-58); WO9217497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J.Immunol. 22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779;(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW: 30820 ™: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_(—)002111.1)Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci. USA99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119;(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 ™: 1 [P] Gene Chromosome: 17p 13.3, Genbank accession No.NP_(—)002552.2)Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035(claim 10); Touchman et al (2000) Genome Res. 10:165-173; WO200222660(claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277(page 82);(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1.359; 359 aa), pI: 8.66, MW: 40225 ™: 1 [P]Gene Chromosome: 9p13.3, Genbank accession No. NP_(—)001773.1)WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877;Strausberg et al (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903;(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pI:6.20, MW: 74147 ™: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_(—)005573.1)US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996)Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822;WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages24-26);(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW:46925 ™: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_(—)443170.1)WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al(2001) Proc. Natl. Acad. Sci. USA 98(17):9772-9777; WO2003089624 (claim8); EP1347046 (claim 1); WO2003089624 (claim 7);(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pI: 6.88 MW: 106468 ™: 1 [P] GeneChromosome: 1q21, Genbank accession No. Human:AF343662, AF343663,AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187,AY358085; Mouse:AK089756, AY158090, AY506558; NP_(—)112571.1WO2003024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem. Biophys.Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG.18B-1-18B-2);(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_(—)057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;Genbank accession No. AF179274; AY358907, CAF85723, CQ782436WO2004074320 (SEQ ID NO 810); JP2004113151 (SEQ ID NOS 2, 4, 8);WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944(pages 69-70); WO200230268 (page 329); WO200190304 (SEQ ID NO 2706);US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350;US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152;Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang etal (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J.Cancer. October 15; 94(2):178-84.

The parent antibody may also be a fusion protein comprising analbumin-binding peptide (ABP) sequence (Dennis et al. (2002) “AlbuminBinding As A General Strategy For Improving The Pharmacokinetics OfProteins” J Biol. Chem. 277:35035-35043; WO 01/45746). Antibodies of theinvention include fusion proteins with ABP sequences taught by: (i)Dennis et al (2002) J Biol. Chem. 277:35035-35043 at Tables III and IV,page 35038; (ii) US 20040001827 at [0076] SEQ ID NOS: 9-22; and (iii) WO01/45746 at pages 12-13, SEQ ID NOS: z1-z14, and all of which areincorporated herein by reference.

Mutagenesis

DNA encoding an amino acid sequence variant of the starting polypeptideis prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may be constructed also byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. Mutagenic primers encode the cysteine codonreplacement(s). Standard mutagenesis techniques can be employed togenerate DNA encoding such mutant cysteine engineered antibodies.General guidance can be found in Sambrook et al Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; and Ausubel et al Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, New York, N.Y., 1993.

Site-directed mutagenesis is one method for preparing substitutionvariants, i.e. mutant proteins. This technique is well known in the art(see for example, Carter (1985) et al Nucleic Acids Res. 13:4431-4443;Ho et al (1989) Gene (Amst.) 77:51-59; and Kunkel et al (1987) Proc.Natl. Acad. Sci. USA 82:488). Briefly, in carrying out site-directedmutagenesis of DNA, the starting DNA is altered by first hybridizing anoligonucleotide encoding the desired mutation to a single strand of suchstarting DNA. After hybridization, a DNA polymerase is used tosynthesize an entire second strand, using the hybridized oligonucleotideas a primer, and using the single strand of the starting DNA as atemplate. Thus, the oligonucleotide encoding the desired mutation isincorporated in the resulting double-stranded DNA. Site-directedmutagenesis may be carried out within the gene expressing the protein tobe mutagenized in an expression plasmid and the resulting plasmid may besequenced to confirm the introduction of the desired cysteinereplacement mutations (Liu et al (1998) J. Biol. Chem. 273:20252-20260).Site-directed of protocols and formats, including those commerciallyavailable, e.g. QuikChangeR Multi Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.).

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, (1990) in PCR Protocols, pp.177-183, Academic Press; Ito et al (1991) Gene 102:67-70; Bernhard et al(1994) Bioconjugate Chem. 5:126-132; and Vallette et al (1989) Nuc.Acids Res. 17:723-733. Briefly, when small amounts of template DNA areused as starting material in a PCR, primers that differ slightly insequence from the corresponding region in a template DNA can be used togenerate relatively large quantities of a specific DNA fragment thatdiffers from the template sequence only at the positions where theprimers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al (1985) Gene 34:315-323. Thestarting material is the plasmid (or other vector) comprising thestarting polypeptide DNA to be mutated. The codon(s) in the starting DNAto be mutated are identified. There must be a unique restrictionendonuclease site on each side of the identified mutation site(s). If nosuch restriction sites exist, they may be generated using the abovedescribed oligonucleotide-mediated mutagenesis method to introduce themat appropriate locations in the starting polypeptide DNA. The plasmidDNA is cut at these sites to linearize it. A double-strandedoligonucleotide encoding the sequence of the DNA between the restrictionsites but containing the desired mutation(s) is synthesized usingstandard procedures, wherein the two strands of the oligonucleotide aresynthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 5′ and 3′ ends that arecompatible with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutatedDNA sequence. Mutant DNA containing the encoded cysteine replacementscan be confirmed by DNA sequencing.

Single mutations are also generated by oligonucleotide directedmutagenesis using double stranded plasmid DNA as template by PCR basedmutagenesis (Sambrook and Russel, (2001) Molecular Cloning: A LaboratoryManual, 3rd edition; Zoller et al (1983) Methods Enzymol. 100:468-500;Zoller, M. J. and Smith, M. (1982) Nucl. Acids Res. 10:6487-6500).

In the present invention, hu4D5Fabv8 displayed on M13 phage (Gerstner etal (2002) “Sequence Plasticity In The Antigen-Binding Site Of ATherapeutic Anti-HER2 Antibody”, J Mol. Biol. 321:851-62) was used forexperiments as a model system. Cysteine mutations were introduced inhu4D5Fabv8-phage, hu4D5Fabv8, and ABP-hu4D5Fabv8 constructs. Thehu4D5-ThioFab-Phage preps were carried out using the polyethylene glycol(PEG) precipitation method as described earlier (Lowman, Henry B. (1998)Methods in Molecular Biology (Totowa, N.J.) 87 (Combinatorial PeptideLibrary Protocols) 249-264).

Oligonucleotides are prepared by the phosphoramidite synthesis method(U.S. Pat. No. 4,415,732; U.S. Pat. No. 4,458,066; Beaucage, S, andIyer, R. (1992) “Advances in the synthesis of oligonucleotides by thephosphoramidite approach”, Tetrahedron 48:2223-2311). Thephosphoramidite method entails cyclical addition of nucleotide monomerunits with a reactive 3′ phosphoramidite moiety to an oligonucleotidechain growing on a solid-support comprised of controlled-pore glass orhighly crosslinked polystyrene, and most commonly in the 3′ to 5′direction in which the 3′ terminus nucleoside is attached to thesolid-support at the beginning of synthesis (U.S. Pat. No. 5,047,524;U.S. Pat. No. 5,262,530). The method is usually practiced usingautomated, commercially available synthesizers (Applied Biosystems,Foster City, Calif.). Oligonucleotides can be chemically labelled withnon-isotopic moieties for detection, capture, stabilization, or otherpurposes (Andrus, A. “Chemical methods for 5′ non-isotopic labelling ofPCR probes and primers” (1995) in PCR 2: A Practical Approach, OxfordUniversity Press, Oxford, pp. 39-54; Hermanson, G. in BioconjugateTechniques (1996) Academic Press, San Diego, pp. 40-55, 643-671; Keller,G. and Manak, M. in DNA Probes Second Edition (1993), Stockton Press,New York, pp. 121-23).

PHESELECTOR Assay

The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) assayallows for detection of reactive cysteine groups in antibodies in anELISA phage format. The process of coating the protein (e.g. antibody)of interest on well surfaces, followed incubation with phage particlesand then HRP labeled secondary antibody with absorbance detection isdetailed in Example 2. Mutant proteins displayed on phage may bescreened in a rapid, robust, and high-throughput manner. Libraries ofcysteine engineered antibodies can be produced and subjected to bindingselection using the same approach to identify appropriately reactivesites of free Cys incorporation from random protein-phage libraries ofantibodies or other proteins. This technique includes reacting cysteinemutant proteins displayed on phage with an affinity reagent or reportergroup which is also thiol-reactive. FIG. 8 illustrates the PHESELECTORAssay by a schematic representation depicting the binding of Fab orThioFab to HER2 (top) and biotinylated ThioFab to streptavidin (bottom).

Protein Expression and Purification

DNA encoding the cysteine engineered antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, orother mammalian host cells, such as myeloma cells (U.S. Pat. No.5,807,715; US 2005/0048572; US 2004/0229310) that do not otherwiseproduce the antibody protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. The yields of hu4D5Fabv8cysteine engineered antibodies were similar to wild type hu4D5Fabv8.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al (1993) Curr. Opinion in Immunol.5:256-262 and Pl{umlaut over (c)}kthun (1992) Immunol. Revs.130:151-188.

After design and selection, cysteine engineered antibodies, e.g.ThioFabs, with highly reactive unpaired Cys residues, may be producedby: (i) expression in a bacterial, e.g. E. coli, system or a mammaliancell culture system (WO 01/00245), e.g. Chinese Hamster Ovary cells(CHO); and (ii) purification using common protein purificationtechniques (Lowman et al (1991) J. Biol. Chem. 266(17):10982-10988).

ThioFabs were expressed upon induction in 34B8, a non-suppressor E. colistrain (Baca et al (1997) Journal Biological Chemistry272(16):10678-84). See Example 3a. The harvested cell pellet wasresuspended in PBS (phosphate buffered saline), total cell lysis wasperformed by passing through a microfluidizer and the ThioFabs werepurified by affinity chromatography with protein G SEPHAROSE™(Amersham). ThioFabs were conjugated with biotin-PEO-maleimide asdescribed above and the biotinylated-ThioFabs were further purified bySuperdex-200™ (Amersham) gel filtration chromatography, which eliminatedthe free biotin-PEO-maleimide and the oligomeric fraction of ThioFabs.

Mass Spectroscopy Analysis

Liquid chromatography electrospray ionization mass spectrometric(LC-ESI-MS) analysis was employed for the accurate molecular weightdetermination of biotin conjugated Fab (Cole, R. B. Electro SprayIonization Mass Spectrometry: Fundamentals, Instrumentation AndApplications. (1997) Wiley, New York). The amino acid sequence ofbiotinylated hu4D5Fabv8 (A121C) peptide was determined by trypticdigestion followed by LC-ESI-Tandem MS analysis (Table 4, Example 3b).

The antibody Fab fragment hu4D5Fabv8 contains about 445 amino acidresidues, including 10 Cys residues (five on the light and five on theheavy chain). The high-resolution structure of the humanized 4D5variable fragment (Fv4D5) has been established, see: Eigenbrot et al“X-Ray Structures Of The Antigen-Binding Domains From Three Variants OfHumanized Anti-P185her2 Antibody 4D5 And Comparison With MolecularModeling” (1993) J Mol. Biol. 229:969-995). All the Cys residues arepresent in the form of disulfide bonds, therefore these residues do nothave any reactive thiol groups to conjugate with drug-maleimide (unlesstreated with a reducing agent). Hence, the newly engineered Cys residue,can remain unpaired, and able to react with, i.e. conjugate to, anelectrophilic linker reagent or drug-linker intermediate, such as adrug-maleimide. FIG. 1A shows a three-dimensional representation of thehu4D5Fabv8 antibody fragment derived by X-ray crystal coordinates. Thestructure positions of the engineered Cys residues of the heavy andlight chains are numbered according to a sequential numbering system.This sequential numbering system is correlated to the Kabat numberingsystem (Kabat et al., (1991) Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md.) for the 4d5v7fabH variant of trastuzumab according toFIG. 1B which shows the sequential numbering scheme (top row), startingat the N-terminus, differs from the Kabat numbering scheme (bottom row)by insertions noted by a,b,c. Using the Kabat numbering system, theactual linear amino acid sequence may contain fewer or additional aminoacids corresponding to a shortening of, or insertion into, a FR or CDRof the variable domain. The cysteine engineered heavy chain variantsites are identified by the sequential numbering and Kabat numberingschemes in the following chart:

4D5Fab Heavy chain variants Sequential Numbering Kabat Numbering A40CAla-40 Ala-40 A88C Ala-88 Ala-84 S119C Ser-119 Ser-112 S120C Ser-120Ser-113 A121C Ala-121 Ala-114 S122C Ser-122 Ser-115 A175C Ala-175Ala-168

M13 phagemid-Cys mutant Fabs (FIGS. 3A and 3B) can be rapidly screenedcompared to Fab proteins. Phagemid-ThioFab binding to antigen and tostreptavidin can be tested by coating HER2 and streptavidin,respectively, onto ELISA plates followed by probing with anti-Fab-HRP(Horse radish peroxidase) as described in Example 2 and depicted in FIG.8. This method allowed simultaneous monitoring of the effect on theantigen binding and the reactivity of the thiol group by the engineeredCys residue/conjugated biotin molecule. Also, the method can be appliedto screen the reactive thiol groups for any protein displayed on M13phage. Conjugated or unconjugated phagemid-ThioFabs are purified bysimple PEG precipitation.

The antigen-binding fragment of humanized 4D5 (hu4D5Fab) is wellexpressed in E. Coli and has been displayed on bacteriophage (Garrard etal (1993) Gene 128: 103-109). The antibody Fab fragment hu4D5Fabv8 wasdisplayed on M13 phage as a model system in the ELISA based assay toprobe thiol reactivity. FIG. 8 is a graphical representation of thePHESELECTOR assay, depicting binding of a biotinylated ThioFab phage andan anti-phage HRP antibody to HER2 (top) and Streptavidin (bottom). Fiveamino acid residues (L-Ala43, H-Ala40, H-Serl 19, H-Ala121 and H-Ser122)were initially selected from crystal structure information as remotefrom the antigen binding surface (Eigenbrot et al. (1993) J Mol Biol.229:969-995). The Protein Database X-ray crystal structure wasdesignated as 1FVC. Cys residues were engineered at these positions bysite directed mutagenesis. ThioFab-phage preparations were isolated andreacted with the biotinylation reagent.

Biotin conjugated and unconjugated variants were tested for HER2 andstreptavidin binding using an ELISA based PHESELECTOR assay (FIG. 8,Example 2) with an HRP (horseradish peroxidase)-conjugated anti-phageantibody. The interaction of non-biotinylated phage-hu4D5Fabv8 (FIG. 2A)and biotinylated phage-hu4D5Fabv8 (FIG. 2B) with BSA (open box), HER2(grey box) or streptavidin (solid box) were monitored throughanti-M13-horseradish peroxidase (HRP) antibody by developing a standardHRP reaction and measuring absorbance at 450 nm. The absorbance producedby turnover of a colorimetric substrate was measured at 450 nm. Thereactivity of ThioFab with HER2 measures antigen binding. The reactivityof ThioFab with streptavidin measures the extent of biotinylation. Thereactivity of ThioFab with BSA is a negative control for nonspecificinteraction. As seen in FIG. 2A, all the ThioFab-phage variants havesimilar binding to HER2 compared to that of wild type hu4D5Fabv8-phage.Furthermore, conjugation with biotin did not interfere in the ThioFabbinding to HER2 (FIG. 2B).

Surprisingly and unexpectedly, the ThioFabs-phage samples showed varyinglevels of streptavidin binding activity. From all the testedphage-ThioFabs, the A121C cysteine engineered antibody exhibited maximalthiol reactivity. Even though wild type hu4D5Fabv8-phage was incubatedwith the same amounts of biotin-maleimide, these phage had littlestreptavidin binding indicating that preexisting cysteine residues(involved in disulfide bond formation) from the hu4D5Fabv8 and M13 phagecoat proteins did not interfere with the site-specific conjugation ofbiotin-maleimide. These results demonstrate that the phage ELISA assaycan be used successfully to screen reactive thiol groups on the Fabsurface.

The PHESELECTOR assay allows screening of reactive thiol groups inantibodies. Identification of the A121C variant by this method isexemplary. The entire Fab molecule may be effectively searched toidentify more ThioFab variants with reactive thiol groups. A parameter,fractional surface accessibility, was employed to identify andquantitate the accessibility of solvent to the amino acid residues in apolypeptide. The surface accessibility can be expressed as the surfacearea (Å²) that can be contacted by a solvent molecule, e.g. water. Theoccupied space of water is approximated as a 1.4 Å radius sphere.Software is freely available or licensable (Secretary to CCP4, DaresburyLaboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825,or by internet: www.ccp4.ac.uk/dist/html/INDEX.html) as the CCP4 Suiteof crystallography programs which employ algorithms to calculate thesurface accessibility of each amino acid of a protein with known x-raycrystallography derived coordinates (“The CCP4 Suite: Programs forProtein Crystallography” (1994) Acta. Cryst. D50:760-763). Two exemplarysoftware modules that perform surface accessibility calculations are“AREAIMOL” and “SURFACE”, based on the algorithms of B. Lee and F. M.Richards (1971) J. Mol. Biol. 55:379-400. AREAIMOL defines the solventaccessible surface of a protein as the locus of the centre of a probesphere (representing a solvent molecule) as it rolls over the Van derWaals surface of the protein. AREAIMOL calculates the solvent accessiblesurface area by generating surface points on an extended sphere abouteach atom (at a distance from the atom centre equal to the sum of theatom and probe radii), and eliminating those that lie within equivalentspheres associated with neighboring atoms. AREAIMOL finds the solventaccessible area of atoms in a PDB coordinate file, and summarizes theaccessible area by residue, by chain and for the whole molecule.Accessible areas (or area differences) for individual atoms can bewritten to a pseudo-PDB output file. AREAIMOL assumes a single radiusfor each element, and only recognizes a limited number of differentelements. Unknown atom types (i.e. those not in AREAIMOL's internaldatabase) will be assigned the default radius of 1.8 Å. The list ofrecognized atoms is:

Atom Atomic no. Van der Waals rad. (Å) C 6 1.80 N 7 1.65 O 8 1.60 Mg 121.60 S 16 1.85 P 15 1.90 Cl 17 1.80 Co 27 1.80

AREAIMOL and SURFACE report absolute accessibilities, i.e. the number ofsquare Angstroms (Å). Fractional surface accessibility is calculated byreference to a standard state relevant for an amino acid within apolypeptide. The reference state is tripeptide Gly-X-Gly, where X is theamino acid of interest, and the reference state should be an ‘extended’conformation, i.e. like those in beta-strands. The extended conformationmaximizes the accessibility of X. A calculated accessible area isdivided by the accessible area in a Gly-X-Gly tripeptide reference stateand reports the quotient, which is the fractional accessibility. Percentaccessibility is fractional accessibility multiplied by 100.

Another exemplary algorithm for calculating surface accessibility isbased on the SOLV module of the program xsae (Broger, C., F.Hoffman-LaRoche, Basel) which calculates fractional accessibility of anamino acid residue to a water sphere based on the X-ray coordinates ofthe polypeptide.

The fractional surface accessibility for every amino acid in hu4D5Fabv7was calculated using the crystal structure information (Eigenbrot et al.(1993) J Mol Biol. 229:969-995). The fractional surface accessibilityvalues for the amino acids of the light chain and heavy chain ofhu4D5Fabv7 are shown in descending order in Table 1.

TABLE 1 hu4D5Fabv7-light chain SER A 202 frac acc = 101.236 GLY A 41frac acc = 90.775 GLY A 157 frac acc = 88.186 ASP A 1 frac acc = 87.743SER A 156 frac acc = 83.742 GLY A 57 frac acc = 81.611 SER A 168 fracacc = 79.680 SER A 56 frac acc = 79.181 LYS A 169 frac acc = 77.591 SERA 60 frac acc = 75.291 THR A 109 frac acc = 74.603 CYS A 214 frac acc =72.021 LYS A 126 frac acc = 71.002 SER A 67 frac acc = 66.694 ARG A 18frac acc = 66.126 ASN A 152 frac acc = 65.415 SER A 127 frac acc =65.345 LYS A 190 frac acc = 65.189 LYS A 145 frac acc = 63.342 GLN A 199frac acc = 62.470 GLU A 143 frac acc = 61.681 GLN A 3 frac acc = 59.976LYS A 188 frac acc = 59.680 ARG A 24 frac acc = 59.458 PHE A 53 frac acc= 58.705 SER A 9 frac acc = 58.446 GLN A 27 frac acc = 57.247 ALA A 153frac acc = 56.538 SER A 203 frac acc = 55.864 LYS A 42 frac acc = 54.730GLY A 16 frac acc = 54.612 LYS A 45 frac acc = 54.464 PRO A 204 frac acc= 53.172 GLU A 213 frac acc = 53.084 ALA A 184 frac acc = 52.556 VAL A15 frac acc = 52.460 SER A 7 frac acc = 51.936 LEU A 154 frac acc =51.525 GLN A 100 frac acc = 51.195 SER A 10 frac acc = 49.907 THR A 5frac acc = 48.879 THR A 206 frac acc = 48.853 ASP A 28 frac acc = 48.758GLY A 68 frac acc = 48.690 THR A 20 frac acc = 48.675 ASP A 122 frac acc= 47.359 PRO A 80 frac acc = 46.984 SER A 52 frac acc = 46.917 SER A 26frac acc = 46.712 TYR A 92 frac acc = 46.218 LYS A 107 frac acc = 45.912GLU A 161 frac acc = 45.100 VAL A 110 frac acc = 44.844 GLU A 81 fracacc = 44.578 PRO A 59 frac acc = 44.290 ASN A 30 frac acc = 42.721 GLN A160 frac acc = 42.692 SER A 114 frac acc = 42.374 PRO A 40 frac acc =41.928 ASP A 151 frac acc = 41.586 SER A 12 frac acc = 40.633 ASN A 210frac acc = 40.158 SER A 63 frac acc = 39.872 ARG A 66 frac acc = 39.669PRO A 8 frac acc = 39.297 SER A 65 frac acc = 39.219 SER A 77 frac acc =38.820 THR A 180 frac acc = 38.296 ASP A 185 frac acc = 38.234 THR A 31frac acc = 38.106 THR A 94 frac acc = 37.452 THR A 93 frac acc = 37.213THR A 197 frac acc = 36.709 SER A 182 frac acc = 36.424 GLY A 128 fracacc = 35.779 LYS A 207 frac acc = 35.638 ASP A 17 frac acc = 35.413 GLYA 200 frac acc = 35.274 GLU A 165 frac acc = 35.067 ALA A 112 frac acc =34.912 GLN A 79 frac acc = 34.601 VAL A 191 frac acc = 33.935 SER A 208frac acc = 33.525 LYS A 39 frac acc = 33.446 GLU A 123 frac acc = 32.486THR A 69 frac acc = 32.276 SER A 76 frac acc = 32.108 HIS A 189 frac acc= 31.984 ARG A 108 frac acc = 31.915 ASN A 158 frac acc = 31.447 VAL A205 frac acc = 31.305 SER A 14 frac acc = 31.094 GLN A 155 frac acc =30.630 GLU A 187 frac acc = 30.328 ARG A 211 frac acc = 30.027 LYS A 183frac acc = 29.751 ASN A 138 frac acc = 29.306 ASP A 170 frac acc =29.041 SER A 159 frac acc = 27.705 GLN A 147 frac acc = 27.485 THR A 22frac acc = 27.121 ALA A 43 frac acc = 26.801 ARG A 142 frac acc = 26.447LEU A 54 frac acc = 25.882 ASP A 167 frac acc = 25.785 THR A 129 fracacc = 23.880 ALA A 144 frac acc = 23.652 VAL A 163 frac acc = 22.261 PROA 95 frac acc = 20.607 ALA A 111 frac acc = 19.942 LYS A 103 frac acc =18.647 LEU A 181 frac acc = 18.312 THR A 72 frac acc = 18.226 GLU A 195frac acc = 18.006 THR A 178 frac acc = 17.499 THR A 85 frac acc = 17.343ASP A 70 frac acc = 17.194 LEU A 11 frac acc = 16.568 PHE A 116 frac acc= 16.406 THR A 97 frac acc = 16.204 ARG A 61 frac acc = 16.192 TYR A 49frac acc = 16.076 SER A 50 frac acc = 15.746 LYS A 149 frac acc = 15.510GLU A 55 frac acc = 14.927 LEU A 201 frac acc = 14.012 GLY A 64 frac acc= 13.735 GLY A 212 frac acc = 13.396 PHE A 98 frac acc = 12.852 THR A 74frac acc = 12.169 SER A 171 frac acc = 11.536 PRO A 141 frac acc =11.073 PHE A 83 frac acc = 10.871 THR A 164 frac acc = 10.325 ALA A 32frac acc = 9.971 HIS A 198 frac acc = 9.958 VAL A 146 frac acc = 9.861SER A 121 frac acc = 9.833 ALA A 13 frac acc = 9.615 GLU A 105 frac acc= 9.416 SER A 162 frac acc = 9.304 ILE A 117 frac acc = 8.780 HIS A 91frac acc = 8.557 ALA A 193 frac acc = 8.547 GLN A 37 frac acc = 8.442VAL A 58 frac acc = 8.281 PRO A 120 frac acc = 8.095 GLN A 38 frac acc =6.643 PRO A 113 frac acc = 6.594 GLY A 101 frac acc = 6.558 TYR A 140frac acc = 5.894 VAL A 115 frac acc = 5.712 TYR A 87 frac acc = 4.539SER A 176 frac acc = 4.106 ILE A 2 frac acc = 4.080 ASN A 137 frac acc =3.906 TRP A 148 frac acc = 3.676 GLY A 99 frac acc = 3.550 PRO A 44 fracacc = 3.543 LEU A 175 frac acc = 3.488 VAL A 19 frac acc = 3.420 ILE A106 frac acc = 3.337 PRO A 119 frac acc = 2.953 LEU A 46 frac acc =2.887 GLN A 6 frac acc = 2.860 TYR A 173 frac acc = 2.825 VAL A 150 fracacc = 2.525 GLN A 166 frac acc = 2.525 THR A 172 frac acc = 2.436 LEU A125 frac acc = 2.398 PRO A 96 frac acc = 2.387 LEU A 47 frac acc = 2.180ALA A 51 frac acc = 1.837 PHE A 118 frac acc = 1.779 PHE A 62 frac acc =1.581 ALA A 25 frac acc = 1.538 VAL A 133 frac acc = 1.315 ASP A 82 fracacc = 1.141 LEU A 179 frac acc = 0.872 GLN A 124 frac acc = 0.787 MET A4 frac acc = 0.778 SER A 177 frac acc = 0.693 SER A 131 frac acc = 0.693LEU A 135 frac acc = 0.654 PHE A 71 frac acc = 0.593 TRP A 35 frac acc =0.448 PHE A 209 frac acc = 0.395 TYR A 186 frac acc = 0.259 LEU A 78frac acc = 0.157 VAL A 196 frac acc = 0.000 VAL A 132 frac acc = 0.000VAL A 104 frac acc = 0.000 VAL A 33 frac acc = 0.000 VAL A 29 frac acc =0.000 TYR A 192 frac acc = 0.000 TYR A 86 frac acc = 0.000 TYR A 36 fracacc = 0.000 THR A 102 frac acc = 0.000 SER A 174 frac acc = 0.000 PHE A139 frac acc = 0.000 LEU A 136 frac acc = 0.000 LEU A 73 frac acc =0.000 ILE A 75 frac acc = 0.000 ILE A 48 frac acc = 0.000 ILE A 21 fracacc = 0.000 GLN A 90 frac acc = 0.000 GLN A 89 frac acc = 0.000 CYS A194 frac acc = 0.000 CYS A 134 frac acc = 0.000 CYS A 88 frac acc =0.000 CYS A 23 frac acc = 0.000 ALA A 130 frac acc = 0.000 ALA A 84 fracacc = 0.000 ALA A 34 frac acc = 0.000 hu4D5Fabv7-heavy chain SER B 179frac acc = 99.479 GLY B 42 frac acc = 95.850 GLU B 1 frac acc = 87.276GLY B 66 frac acc = 84.541 ASP B 102 frac acc = 83.794 SER B 75 frac acc= 80.567 GLY B 140 frac acc = 80.344 ASN B 211 frac acc = 79.588 GLY B197 frac acc = 78.676 ASP B 62 frac acc = 77.716 GLY B 103 frac acc =77.176 SER B 163 frac acc = 76.664 SER B 139 frac acc = 74.946 LYS B 213frac acc = 74.442 ALA B 165 frac acc = 74.339 THR B 167 frac acc =73.934 SER B 122 frac acc = 72.870 SER B 194 frac acc = 71.959 PRO B 41frac acc = 71.540 THR B 198 frac acc = 68.668 SER B 222 frac acc =68.128 LYS B 43 frac acc = 67.782 GLY B 26 frac acc = 67.782 THR B 138frac acc = 65.826 ASP B 31 frac acc = 64.222 GLY B 15 frac acc = 64.172SER B 168 frac acc = 62.100 SER B 120 frac acc = 61.332 LYS B 76 fracacc = 61.092 GLY B 141 frac acc = 59.419 SER B 137 frac acc = 59.179 TYRB 57 frac acc = 58.916 GLU B 89 frac acc = 58.483 SER B 180 frac acc =56.289 LYS B 65 frac acc = 55.044 ASP B 215 frac acc = 54.656 GLN B 13frac acc = 53.719 GLN B 112 frac acc = 53.215 TYR B 105 frac acc =51.940 ALA B 88 frac acc = 51.602 GLY B 164 frac acc = 50.259 PRO B 192frac acc = 49.826 THR B 158 frac acc = 49.694 THR B 142 frac acc =48.896 ASN B 55 frac acc = 48.344 LYS B 136 frac acc = 48.312 ARG B 19frac acc = 48.082 PRO B 156 frac acc = 47.366 PRO B 174 frac acc =47.157 LYS B 217 frac acc = 47.102 GLN B 199 frac acc = 46.650 SER B 17frac acc = 45.980 SER B 85 frac acc = 45.824 PRO B 14 frac acc = 45.729THR B 54 frac acc = 45.503 THR B 200 frac acc = 45.369 LEU B 177 fracacc = 45.337 GLY B 8 frac acc = 44.898 SER B 7 frac acc = 43.530 THR B69 frac acc = 43.503 PRO B 220 frac acc = 43.378 LYS B 208 frac acc =43.138 LYS B 30 frac acc = 42.380 ALA B 23 frac acc = 41.952 GLU B 46frac acc = 41.430 SER B 25 frac acc = 41.323 ARG B 87 frac acc = 41.282LYS B 124 frac acc = 40.888 ASN B 28 frac acc = 40.529 GLN B 3 frac acc= 39.824 THR B 123 frac acc = 39.306 SER B 63 frac acc = 38.867 GLY B 56frac acc = 38.582 GLY B 169 frac acc = 38.469 THR B 172 frac acc =38.421 PRO B 209 frac acc = 38.309 GLY B 101 frac acc = 38.040 TYR B 109frac acc = 36.829 LYS B 221 frac acc = 36.520 GLY B 44 frac acc = 35.147GLY B 181 frac acc = 34.735 THR B 58 frac acc = 34.457 GLY B 9 frac acc= 34.254 VAL B 5 frac acc = 34.198 ALA B 121 frac acc = 33.049 SER B 127frac acc = 32.390 GLY B 10 frac acc = 32.230 SER B 71 frac acc = 30.659ASP B 73 frac acc = 30.245 LEU B 115 frac acc = 29.867 LEU B 11 frac acc= 29.825 ASN B 84 frac acc = 29.765 SER B 210 frac acc = 28.656 GLU B155 frac acc = 28.162 SER B 160 frac acc = 26.526 CYS B 223 frac acc =26.270 GLY B 16 frac acc = 26.158 ILE B 202 frac acc = 26.068 GLN B 82frac acc = 25.836 SER B 193 frac acc = 25.550 ASN B 77 frac acc = 25.418ARG B 59 frac acc = 25.301 VAL B 93 frac acc = 25.254 THR B 74 frac acc= 24.902 GLU B 219 frac acc = 24.778 ASN B 206 frac acc = 24.647 VAL B170 frac acc = 24.549 TYR B 52 frac acc = 24.298 ALA B 175 frac acc =23.804 LYS B 216 frac acc = 23.277 VAL B 214 frac acc = 23.150 GLY B 125frac acc = 22.802 ASN B 162 frac acc = 22.245 ALA B 72 frac acc = 22.166ALA B 40 frac acc = 21.974 LEU B 18 frac acc = 20.273 THR B 212 frac acc= 20.170 LEU B 182 frac acc = 19.619 TYR B 33 frac acc = 19.398 THR B190 frac acc = 19.365 VAL B 176 frac acc = 18.941 SER B 21 frac acc =18.929 SER B 119 frac acc = 18.877 THR B 91 frac acc = 18.237 ASP B 151frac acc = 17.849 THR B 114 frac acc = 17.601 SER B 134 frac acc =17.571 LEU B 196 frac acc = 17.090 TYR B 60 frac acc = 16.575 TYR B 183frac acc = 15.968 VAL B 2 frac acc = 15.901 PRO B 130 frac acc = 15.342LEU B 166 frac acc = 15.268 GLY B 100 frac acc = 15.003 PHE B 27 fracacc = 14.383 ASN B 204 frac acc = 13.873 PHE B 104 frac acc = 13.836 TYRB 80 frac acc = 13.490 VAL B 159 frac acc = 12.782 ARG B 67 frac acc =12.362 GLN B 178 frac acc = 12.131 HIS B 171 frac acc = 11.412 SER B 184frac acc = 11.255 ARG B 98 frac acc = 11.115 PRO B 53 frac acc = 11.071GLN B 39 frac acc = 11.037 SER B 195 frac acc = 10.909 ASP B 108 fracacc = 10.525 LEU B 185 frac acc = 10.464 GLY B 113 frac acc = 10.406 THRB 78 frac acc = 10.213 THR B 117 frac acc = 9.990 LYS B 150 frac acc =9.447 VAL B 157 frac acc = 9.323 VAL B 12 frac acc = 9.207 TRP B 110frac acc = 9.069 ALA B 143 frac acc = 8.903 SER B 135 frac acc = 8.897PHE B 129 frac acc = 8.895 ARG B 50 frac acc = 8.639 ALA B 61 frac acc =8.547 ALA B 132 frac acc = 7.882 VAL B 191 frac acc = 7.366 PRO B 126frac acc = 7.258 PHE B 153 frac acc = 6.918 PRO B 154 frac acc = 6.767PRO B 133 frac acc = 6.767 TRP B 99 frac acc = 6.502 THR B 32 frac acc =6.291 LEU B 45 frac acc = 4.649 VAL B 128 frac acc = 4.515 ILE B 51 fracacc = 4.307 SER B 186 frac acc = 4.084 PHE B 173 frac acc = 3.969 ARG B38 frac acc = 3.734 TRP B 47 frac acc = 3.561 VAL B 118 frac acc = 3.409ALA B 24 frac acc = 3.376 TYR B 95 frac acc = 3.242 GLU B 6 frac acc =3.216 ALA B 144 frac acc = 3.167 ILE B 70 frac acc = 1.958 GLY B 111frac acc = 1.868 LEU B 4 frac acc = 1.808 TYR B 201 frac acc = 1.758 LEUB 148 frac acc = 1.744 PHE B 68 frac acc = 1.708 VAL B 188 frac acc =1.315 CYS B 22 frac acc = 0.935 TRP B 161 frac acc = 0.876 LEU B 131frac acc = 0.654 VAL B 205 frac acc = 0.495 ALA B 92 frac acc = 0.356ALA B 79 frac acc = 0.356 VAL B 64 frac acc = 0.263 ILE B 29 frac acc =0.227 VAL B 218 frac acc = 0.000 VAL B 189 frac acc = 0.000 VAL B 149frac acc = 0.000 VAL B 116 frac acc = 0.000 VAL B 48 frac acc = 0.000VAL B 37 frac acc = 0.000 TYR B 152 frac acc = 0.000 TYR B 94 frac acc =0.000 TRP B 36 frac acc = 0.000 SER B 187 frac acc = 0.000 SER B 97 fracacc = 0.000 MET B 107 frac acc = 0.000 MET B 83 frac acc = 0.000 LEU B145 frac acc = 0.000 LEU B 86 frac acc = 0.000 LEU B 81 frac acc = 0.000LEU B 20 frac acc = 0.000 ILE B 34 frac acc = 0.000 HIS B 207 frac acc =0.000 HIS B 35 frac acc = 0.000 GLY B 146 frac acc = 0.000 CYS B 203frac acc = 0.000 CYS B 147 frac acc = 0.000 CYS B 96 frac acc = 0.000ASP B 90 frac acc = 0.000 ALA B 106 frac acc = 0.000 ALA B 49 frac acc =0.000

The following two criteria were applied to identify the residues ofhu4D5Fabv8 that can be engineered to replace with Cys residues:

1. Amino acid residues that are completely buried are eliminated, i.e.less than 10% fractional surface accessibility. Table 1 shows there are134 (light chain) and 151 (heavy chain) residues of hu4D5Fabv8 that aremore than 10% accessible (fractional surface accessibility). The top tenmost accessible Ser, Ala and Val residues were selected due to theirclose structural similarity to Cys over other amino acids, introducingonly minimal structural constraints in the antibody by newly engineeredCys. Other cysteine replacement sites can also be screened, and may beuseful for conjugation.

2. Residues are sorted based on their role in functional and structuralinteractions of Fab. The residues which are not involved in antigeninteractions and distant from the existing disulfide bonds were furtherselected. The newly engineered Cys residues should be distinct from, andnot interfere with, antigen binding nor mispair with cysteines involvedin disulfide bond formation.

The following residues of hu4D5Fabv8 possessed the above criteria andwere selected to be replaced with Cys: L-V15, L-A43, L-V110, L-A144,L-S168, H-A88, H-A121, H-S122, H-A175 and H-S179 (shown in FIG. 1).

Thiol reactivity may be generalized to any antibody where substitutionof amino acids with reactive cysteine amino acids may be made within theranges in the light chain selected from: L-10 to L-20; L-38 to L-48;L-105 to L-115; L-139 to L-149; L-163 to L-173; and within the ranges inthe heavy chain selected from: H-35 to H-45; H-83 to H-93; H-114 toH-127; and H-170 to H-184, and in the Fc region within the rangesselected from H-268 to H-291; H-319 to H-344; H-370 to H-380; and H-395to H-405.

Thiol reactivity may also be generalized to certain domains of anantibody, such as the light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. Cysteine replacements resulting inthiol reactivity values of 0.6 and higher may be made in the heavy chainconstant domains α, δ, ε, γ, and μ of intact antibodies: IgA, IgD, IgE,IgG, and IgM, respectively, including the IgG subclasses: IgG1, IgG2,IgG3, IgG4, IgA, and IgA2.

It is evident from the crystal structure data that the selected 10 Cysmutants are far away from the antigen-combining site, such as theinterface with HER2 in this case. These mutants can be testedexperimentally for indirect effects on functional interactions. Thethiol reactivities of all the Cys Fab variants were measured andcalculated as described in Examples 1 and 2, and presented in Table 2.The residues L-V15C, L-V110C, H-A88C and H-A121C have reactive andstable thiol groups (FIGS. 3A and 3B). Mutants V15C, V110C, A144C, S168Care light chain Cysvariants. Mutants A88C, A121C, A175C, S179C are heavychain Cys variants. It was surprising and unexpected that the sites withhigh fractional surface accessibility did not have the highest thiolreactivity as calculated by the PHESELECTOR assay (Table 2). In otherwords, fractional surface accessibility (Tables 1, 2) did not correlatewith thiol reactivity (Table 2). In fact, the Cys residues engineered atthe sites with moderate surface accessibility of 20% to 80% (FIG. 4A,Table 1), or partially exposed sites, like Ala or Val residues,exhibited better thiol reactivity, i.e. >0.6, (FIG. 3B, Table 2) thanthe Cys introduced at Ser residues, thus necessitating the use ofPHESELECTOR assay in the screening of thiol reactive sites since thecrystal structure information alone is not sufficient to select thesesites (FIGS. 3B and 4A).

Thiol reactivity data is shown in FIGS. 3A and 3B for amino acidresidues of 4D5 ThioFab Cys mutants: (3A) non-biotinylated (control) and(3B) biotinylated phage-ThioFabs. Reactive thiol groups on antibody/Fabsurface were identified by PHESELECTOR assay analyses for theinteraction of non-biotinylated phage-hu4D5Fabv8 (3A) and biotinylatedphage-hu4D5Fabv8 (3B) with BSA (open box), HER2 (grey box) orstreptavidin (solid box). The assay was carried out as described inExample 2. Light chain variants are on the left side and heavy chainvariants are on the right side. The binding of non-biotinylated 4D5ThioFab Cys mutants is low as expected, but strong binding to HER2 isretained. The ratio of binding to streptavidin and to HER2 of thebiotinylated 4D5 ThioFab Cys mutants gives the thiol reactivity valuesin Table 2. Background absorbance at 450 nm or small amounts ofnon-specific protein binding of the biotinylated 4D5 ThioFab Cys mutantsto BSA is also evident in FIG. 3B. Fractional Surface Accessibilityvalues of the selected amino acid residues that were replaced with a Cysresidue are shown in FIG. 4A. Fractional surface accessibility wascalculated from the available hu4D5Fabv7 structure and shown on Table 1(Eigenbrot et al. (1993) J Mol Biol. 229:969-995). The conformationalparameters of the hu4D5Fabv7 and hu4D5Fabv8 structures are highlyconsistent and allow for determination of any correlation betweenfractional surface accessibility calculations of hu4D5Fabv7 and thiolreactivity of hu4D5Fabv8 cysteine mutants. The measured thiol reactivityof phage ThioFab Cys residues introduced at partially exposed residues(Ala or Val) have better thiol reactivity compared to the onesintroduced at Ser residues (Table 2). It can be seen from the ThioFabCys mutants of Table 2 that there is little or no correlation betweenthio reactivity values and fractional surface accessibility.

Amino acids at positions L-15, L-43, L-110, L-144, L-168, H-40, H-88,H-119, H-121, H-122, H-175, and H-179 of an antibody may generally bemutated (replaced) with free cysteine amino acids. Ranges within about 5amino acid residues on each side of these positions may also be replacedwith free cysteine acids, i.e. L-10 to L-20; L-38 to L-48; L-105 toL-115; L-139 to L-149; L-163 to L-173; H-35 to H-45; H-83 to H-93; H-114to H-127; and H-170 to H-184, as well as the ranges in the Fc regionselected from H-268 to H-291; H-319 to H-344; H-370 to H-380; and H-395to H-405, to yield the cysteine engineered antibodies of the invention.

TABLE 2 Thiol reactivity of phage-ThioFabs Fractional SurfacePhage-ThioFab Thiol Accessibility (%) construct Reactivity* (fromTable 1) hu4D5Fabv8-wt 0.125 — L-V15C 0.934 52.46 L-A43C 0.385 26.80L-V110C 0.850 44.84 L-A144C 0.373 23.65 L-S168C 0.514 79.68 H-A40C 0.45021.97 H-A88C 0.914 51.60 H-S119C 0.680 18.88 H-A121C 0.925 33.05 H-S122C0.720 72.87 H-A175C 0.19 23.80 H-S179C 0.446 99.48 L = light chain, H =heavy chain, A = alanine, S = serine, V = valine, C = cysteine *Thiolreactivity is measured as the ratio of OD_(450 nm) for streptavidinbinding to OD_(450 nm) for HER2 (antibody) binding (Example 2). Thiolreactivity value of 1 indicates complete biotinylation of the cysteinethiol.

Two Cys variants from light chain (L-V15C and L-V110C) and two fromheavy chain (H-A88C and H-A121C) were selected for further analysis asthese variants showed the highest thiol reactivity (Table 2).

Unlike phage purification, Fab preparation may require 2-3 days,depending on the scale of production. During this time, thiol groups maylose reactivity due to oxidation. To probe the stability of thiol groupson hu4D5Fabv8-phage, stability of the thiol reactivity of phage-thioFabswas measured (FIG. 4B). After ThioFab-phage purification, on day 1, day2 and day 4, all the samples were conjugated with biotin-PEO-maleimideand probed with phage ELISA assay (PHESELECTOR) to test HER2 andstreptavidin binding. L-V15C, L-V110C, H-A88C and H-A121C retainsignificant amounts of thiol reactivity compared to other ThioFabvariants (FIG. 4B).

Labelled Cysteine Engineered Antibodies

The cysteine engineered antibodies of the invention may be conjugatedwith any label moiety which can be covalently attached to the antibodythrough a reactive cysteine thiol group (Singh et al (2002) Anal.Biochem. 304:147-15; Harlow E. and Lane, D. (1999) Using Antibodies: ALaboratory Manual, Cold Springs Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Lundblad R. L. (1991) Chemical Reagents for ProteinModification, 2nd ed. CRC Press, Boca Raton, Fla.). The attached labelmay function to: (i) provide a detectable signal; (ii) interact with asecond label to modify the detectable signal provided by the first orsecond label, e.g. to give FRET (fluorescence resonance energytransfer); (iii) stabilize interactions or increase affinity of binding,with antigen or ligand; (iv) affect mobility, e.g. electrophoreticmobility or cell-permeability, by charge, hydrophobicity, shape, orother physical parameters, or (v) provide a capture moiety, to modulateligand affinity, antibody/antigen binding, or ionic complexation.

Labelled cysteine engineered antibodies may be useful in diagnosticassays, e.g., for detecting expression of an antigen of interest inspecific cells, tissues, or serum. For diagnostic applications, theantibody will typically be labeled with a detectable moiety. Numerouslabels are available which can be generally grouped into the followingcategories:

(a) Radioisotopes (radionuclides), such as ³H, ¹¹C ¹⁴C, ¹⁸F, ³²P, ³⁵S,⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹³³Xe, ¹⁷⁷Lu,²¹¹At, or ²¹³Bi. Radioisotope labelled antibodies are useful in receptortargeted imaging experiments. The antibody can be labeled with ligandreagents that bind, chelate or otherwise complex a radioisotope metalwhere the reagent is reactive with the engineered cysteine thiol of theantibody, using the techniques described in Current Protocols inImmunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, NewYork, N.Y., Pubs. (1991). Chelating ligands which may complex a metalion include DOTA, DOTP, DOTMA, DTPA and TETA (Macrocyclics, Dallas,Tex.). Radionuclides can be targetted via complexation with theantibody-drug conjugates of the invention (Wu et al (2005) NatureBiotechnology 23(9):1137-1146).

Metal-chelate complexes suitable as antibody labels for imagingexperiments are disclosed: U.S. Pat. No. 5,342,606; U.S. Pat. No.5,428,155; U.S. Pat. No. 5,316,757; U.S. Pat. No. 5,480,990; U.S. Pat.No. 5,462,725; U.S. Pat. No. 5,428,139; U.S. Pat. No. 5,385,893; U.S.Pat. No. 5,739,294; U.S. Pat. No. 5,750,660; U.S. Pat. No. 5,834,456;Hnatowich et al (1983) J. Immunol. Methods 65:147-157; Meares et al(1984) Anal. Biochem. 142:68-78; Mirzadeh et al (1990) BioconjugateChem. 1:59-65; Meares et al (1990) J. Cancer1990, Suppl. 10:21-26; Izardet al (1992) Bioconjugate Chem. 3:346-350; Nikula et al (1995) Nucl.Med. Biol. 22:387-90; Camera et al (1993) Nucl. Med. Biol. 20:955-62;Kukis et al (1998) J. Nucl. Med. 39:2105-2110; Verel et al (2003) J.Nucl. Med. 44:1663-1670; Camera et al (1994) J. Nucl. Med. 21:640-646;Ruegg et al (1990) Cancer Res. 50:4221-4226; Verel et al (2003) J. Nucl.Med. 44:1663-1670; Lee et al (2001) Cancer Res. 61:4474-4482; Mitchell,et al (2003) J. Nucl. Med. 44:1105-1112; Kobayashi et al (1999)Bioconjugate Chem. 10:103-111; Miederer et al (2004) J. Nucl. Med.45:129-137; DeNardo et al (1998) Clinical Cancer Research 4:2483-90;Blend et al (2003) Cancer Biotherapy & Radiopharmaceuticals 18:355-363;Nikula et al (1999) J. Nucl. Med. 40:166-76; Kobayashi et al (1998) J.Nucl. Med. 39:829-36; Mardirossian et al (1993) Nucl. Med. Biol.20:65-74; Roselli et al (1999) Cancer Biotherapy & Radiopharmaceuticals,14:209-20.

(b) Fluorescent labels such as rare earth chelates (europium chelates),fluorescein types including FITC₁₋₅-carboxyfluorescein, 6-carboxyfluorescein; rhodamine types including TAMRA; dansyl; Lissamine;cyanines; phycoerythrins; Texas Red; and analogs thereof. Thefluorescent labels can be conjugated to antibodies using the techniquesdisclosed in Current Protocols in Immunology, supra, for example.Fluorescent dyes and fluorescent label reagents include those which arecommercially available from Invitrogen/Molecular Probes (Eugene, Oreg.)and Pierce Biotechnology, Inc. (Rockford, Ill.).

(c) Various enzyme-substrate labels are available or disclosed (U.S.Pat. No. 4,275,149). The enzyme generally catalyzes a chemicalalteration of a chromogenic substrate that can be measured using varioustechniques. For example, the enzyme may catalyze a color change in asubstrate, which can be measured spectrophotometrically. Alternatively,the enzyme may alter the fluorescence or chemiluminescence of thesubstrate. Techniques for quantifying a change in fluorescence aredescribed above. The chemiluminescent substrate becomes electronicallyexcited by a chemical reaction and may then emit light which can bemeasured (using a chemiluminometer, for example) or donates energy to afluorescent acceptor. Examples of enzymatic labels include luciferases(e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP),alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Techniques for conjugating enzymes to antibodies are described inO'Sullivan et al (1981) “Methods for the Preparation of Enzyme-AntibodyConjugates for use in Enzyme Immunoassay”, in Methods in Enzym. (ed J.Langone & H. Van Vunakis), Academic Press, New York, 73:147-166.

Examples of enzyme-substrate combinations include, for example:

(i) Horseradish peroxidase (HRP) with hydrogen peroxidase as asubstrate,

wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.,orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethylbenzidinehydrochloride (TMB));

(ii) alkaline phosphatase (AP) with para-nitrophenyl phosphate aschromogenic substrate; and

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review, see U.S. Pat. No. 4,275,149and U.S. Pat. No. 4,318,980.

A label may be indirectly conjugated with a cysteine engineeredantibody. For example, the antibody can be conjugated with biotin andany of the three broad categories of labels mentioned above can beconjugated with avidin or streptavidin, or vice versa. Biotin bindsselectively to streptavidin and thus, the label can be conjugated withthe antibody in this indirect manner. Alternatively, to achieve indirectconjugation of the label with the polypeptide variant, the polypeptidevariant is conjugated with a small hapten (e.g., digoxin) and one of thedifferent types of labels mentioned above is conjugated with ananti-hapten polypeptide variant (e.g., anti-digoxin antibody). Thus,indirect conjugation of the label with the polypeptide variant can beachieved (Hermanson, G. (1996) in Bioconjugate Techniques AcademicPress, San Diego).

The polypeptide variant of the present invention may be employed in anyknown assay method, such as ELISA, competitive binding assays, directand indirect sandwich assays, and immunoprecipitation assays (Zola,(1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRCPress, Inc.).

A detection label may be useful for localizing, visualizing, andquantitating a binding or recognition event. The labelled antibodies ofthe invention can detect cell-surface receptors. Another use fordetectably labelled antibodies is a method of bead-based immunocapturecomprising conjugating a bead with a fluorescent labelled antibody anddetecting a fluorescence signal upon binding of a ligand. Similarbinding detection methodologies utilize the surface plasmon resonance(SPR) effect to measure and detect antibody-antigen interactions.

Detection labels such as fluorescent dyes and chemiluminescent dyes(Briggs et al (1997) “Synthesis of Functionalised Fluorescent Dyes andTheir Coupling to Amines and Amino Acids,” J. Chem. Soc., Perkin-Trans.1:1051-1058) provide a detectable signal and are generally applicablefor labelling antibodies, preferably with the following properties: (i)the labelled antibody should produce a very high signal with lowbackground so that small quantities of antibodies can be sensitivelydetected in both cell-free and cell-based assays; and (ii) the labelledantibody should be photostable so that the fluorescent signal may beobserved, monitored and recorded without significant photo bleaching.For applications involving cell surface binding of labelled antibody tomembranes or cell surfaces, especially live cells, the labels preferably(iii) have good water-solubility to achieve effective conjugateconcentration and detection sensitivity and (iv) are non-toxic to livingcells so as not to disrupt the normal metabolic processes of the cellsor cause premature cell death.

Direct quantification of cellular fluorescence intensity and enumerationof fluorescently labelled events, e.g. cell surface binding ofpeptide-dye conjugates may be conducted on an system (FMAT® 8100 HTSSystem, Applied Biosystems, Foster City, Calif.) that automatesmix-and-read, non-radioactive assays with live cells or beads (Miraglia,“Homogeneous cell- and bead-based assays for high throughput screeningusing fluorometric microvolume assay technology”, (1999) J. ofBiomolecular Screening 4:193-204). Uses of labelled antibodies alsoinclude cell surface receptor binding assays, immunocapture assays,fluorescence linked immunosorbent assays (FLISA), caspase-cleavage(Zheng, “Caspase-3 controls both cytoplasmic and nuclear eventsassociated with Fas-mediated apoptosis in vivo”, (1998) Proc. Natl.Acad. Sci. USA 95:618-23; U.S. Pat. No. 6,372,907), apoptosis (Vermes,“A novel assay for apoptosis. Flow cytometric detection ofphosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V” (1995) J. Immunol. Methods 184:39-51) andcytotoxicity assays. Fluorometric microvolume assay technology can beused to identify the up or down regulation by a molecule that istargeted to the cell surface (Swartzman, “A homogeneous and multiplexedimmunoassay for high-throughput screening using fluorometric microvolumeassay technology”, (1999) Anal. Biochem. 271:143-51).

Labelled cysteine engineered antibodies of the invention are useful asimaging biomarkers and probes by the various methods and techniques ofbiomedical and molecular imaging such as: (i) MRI (magnetic resonanceimaging); (ii) MicroCT (computerized tomography); (iii) SPECT (singlephoton emission computed tomography); (iv) PET (positron emissiontomography) Chen et al (2004) Bioconjugate Chem. 15:41-49; (v)bioluminescence; (vi) fluorescence; and (vii) ultrasound.Immunoscintigraphy is an imaging procedure in which antibodies labeledwith radioactive substances are administered to an animal or humanpatient and a picture is taken of sites in the body where the antibodylocalizes (U.S. Pat. No. 6,528,624). Imaging biomarkers may beobjectively measured and evaluated as an indicator of normal biologicalprocesses, pathogenic processes, or pharmacological responses to atherapeutic intervention. Biomarkers may be of several types: Type 0 arenatural history markers of a disease and correlate longitudinally withknown clinical indices, e.g. MRI assessment of synovial inflammation inrheumatoid arthritis; Type I markers capture the effect of anintervention in accordance with a mechanism-of-action, even though themechanism may not be associated with clinical outcome; Type II markersfunction as surrogate endpoints where the change in, or signal from, thebiomarker predicts a clinical benefit to “validate” the targetedresponse, such as measured bone erosion in rheumatoid arthritis by CT.Imaging biomarkers thus can provide pharmacodynamic (PD) therapeuticinformation about: (i) expression of a target protein, (ii) binding of atherapeutic to the target protein, i.e. selectivity, and (iii) clearanceand half-life pharmacokinetic data. Advantages of in vivo imagingbiomarkers relative to lab-based biomarkers include: non-invasivetreatment, quantifiable, whole body assessment, repetitive dosing andassessment, i.e. multiple time points, and potentially transferableeffects from preclinical (small animal) to clinical (human) results. Forsome applications, bioimaging supplants or minimizes the number ofanimal experiments in preclinical studies.

Radionuclide imaging labels include radionuclides such as ³H, ¹¹C, ¹⁴C,¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,¹³³Xe, ¹⁷⁷Lu, ²¹¹At, or ²¹³Bi The radionuclide metal ion can becomplexed with a chelating linker such as DOTA. Linker reagents such asDOTA-maleimide (4-maleimidobutyramidobenzyl-DOTA) can be prepared by thereaction of aminobenzyl-DOTA with 4-maleimidobutyric acid (Fluka)activated with isopropylchloroformate (Aldrich), following the procedureof Axworthy et al (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807).DOTA-maleimide reagents react with the free cysteine amino acids of thecysteine engineered antibodies and provide a metal complexing ligand onthe antibody (Lewis et al (1998) Bioconj. Chem. 9:72-86). Chelatinglinker labelling reagents such as DOTA-NHS(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) are commercially available (Macrocyclics,Dallas, Tex.). Receptor target imaging with radionuclide labelledantibodies can provide a marker of pathway activation by detection andquantitation of progressive accumulation of antibodies in tumor tissue(Albert et al (1998) Bioorg. Med. Chem. Lett. 8:1207-1210). Theconjugated radio-metals may remain intracellular following lysosomaldegradation.

Peptide labelling methods are well known. See Haugland, 2003, MolecularProbes Handbook of Fluorescent Probes and Research Chemicals, MolecularProbes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997)Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Glazer et al (1975) ChemicalModification of Proteins. Laboratory Techniques in Biochemistry andMolecular Biology (T. S. Work and E. Work, Eds.) American ElsevierPublishing Co., New York; Lundblad, R. L. and Noyes, C. M. (1984)Chemical Reagents for Protein Modification, Vols. I and II, CRC Press,New York; Pfleiderer, G. (1985) “Chemical Modification of Proteins”,Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter,Berlin and New York; and Wong (1991) Chemistry of Protein Conjugationand Cross-linking, CRC Press, Boca Raton, Fla.); De Leon-Rodriguez et al(2004) Chem. Eur. J. 10: 1149-1155; Lewis et al (2001) BioconjugateChem. 12:320-324; Li et al (2002) Bioconjugate Chem. 13:110-115; Mier etal (2005) Bioconjugate Chem. 16:240-237.

Peptides and proteins labelled with two moieties, a fluorescent reporterand quencher in sufficient proximity undergo fluorescence resonanceenergy transfer (FRET). Reporter groups are typically fluorescent dyesthat are excited by light at a certain wavelength and transfer energy toan acceptor, or quencher, group, with the appropriate Stokes shift foremission at maximal brightness. Fluorescent dyes include molecules withextended aromaticity, such as fluorescein and rhodamine, and theirderivatives. The fluorescent reporter may be partially or significantlyquenched by the quencher moiety in an intact peptide. Upon cleavage ofthe peptide by a peptidase or protease, a detectable increase influorescence may be measured (Knight, C. (1995) “Fluorimetric Assays ofProteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

The labelled antibodies of the invention may also be used as an affinitypurification agent. In this process, the labelled antibody isimmobilized on a solid phase such a Sephadex resin or filter paper,using methods well known in the art. The immobilized antibody iscontacted with a sample containing the antigen to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except the antigento be purified, which is bound to the immobilized polypeptide variant.Finally, the support is washed with another suitable solvent, such asglycine buffer, pH 5.0, that will release the antigen from thepolypeptide variant.

Labelling reagents typically bear reactive functionality which may react(i) directly with a cysteine thiol of a cysteine engineered antibody toform the labelled antibody, (ii) with a linker reagent to form alinker-label intermediate, or (iii) with a linker antibody to form thelabelled antibody. Reactive functionality of labelling reagents include:maleimide, haloacetyl, iodoacetamide succinimidyl ester (e.g. NHS,N-hydroxysuccinimide), isothiocyanate, sulfonyl chloride,2,6-dichlorotriazinyl, pentafluorophenyl ester, and phosphoramidite,although other functional groups can also be used.

An exemplary reactive functional group is N-hydroxysuccinimidyl ester(NHS) of a carboxyl group substituent of a detectable label, e.g. biotinor a fluorescent dye. The NHS ester of the label may be preformed,isolated, purified, and/or characterized, or it may be formed in situand reacted with a nucleophilic group of an antibody. Typically, thecarboxyl form of the label is activated by reacting with somecombination of a carbodiimide reagent, e.g. dicyclohexylcarbodiimide,diisopropylcarbodiimide, or a uronium reagent, e.g. TSTU(O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate),or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the label. Insome cases, the label and the antibody may be coupled by in situactivation of the label and reaction with the antibody to form thelabel-antibody conjugate in one step. Other activating and couplingreagents include TBTU(2-(1H-benzotriazo-1-yl)-1-1,3,3-tetramethyluroniumhexafluorophosphate), TFFH(N,N′,N″,N′″-tetramethyluronium2-fluoro-hexafluorophosphate), PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, EEDQ(2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline), DCC(dicyclohexylcarbodiimide); DIPCDI (diisopropylcarbodiimide), MSNT(1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole, and aryl sulfonylhalides, e.g. triisopropylbenzenesulfonyl chloride.

Conjugation of Biotin-maleimide to Thiofabs

The above-described ThioFab properties were established in the presenceof phage because fusion of the Fab to the phage coat protein couldpotentially alter Cys thiol accessibility or reactivity. Therefore, theThioFab constructs were cloned into an expression vector under alkalinephosphatase promoter (Chang et al (1987) Gene 55:189-196) and theThioFab expression was induced by growing E. coli cells in thephosphate-free medium. ThioFabs were purified on a Protein G SEPHAROSE™column and analyzed on reducing and non-reducing SDS-PAGE gels. Theseanalyses allow assessment of whether ThioFabs retained their reactivethiol group or were rendered inactive by forming intramolecular orintermolecular disulfide bonds. ThioFabs L-V15C, L-V110C, H-A88C, andH-A121C were expressed and purified by Protein-G SEPHAROSE™ columnchromatography (see methods sections for details). Purified proteinswere analyzed on SDS-PAGE gel in reducing (with DTT) and non-reducing(without DTT) conditions. Other reducing agents such as BME(beta-mercaptoethanol) can used in the gel to cleave interchaindisulfide groups. It is evident from SDS-PAGE gel analysis that themajor (90%) fraction of ThioFab is in the monomeric form, while wildtype hu4D5Fabv8 is essentially in the monomeric form (47 kDa).

ThioFab (A121C) and wild type hu4D5Fabv8 were incubated with 100 foldexcess of biotin-maleimide for 3 hours at room temperature and thebiotinylated Fabs were loaded onto a Superdex-200™ gel filtrationcolumn. This purification step was useful in separating monomeric Fabfrom oligomeric Fab and also from excess free biotin-maleimide (or freecytotoxic drug).

FIG. 5 shows validation of the properties of ThioFab variants in theabsence of the phage context. The proteins without phage fusion,hu4D5Fabv8 and hu4D5Fabv8-A121C (ThioFab-A121C), were expressed andpurified using protein-G agarose beads followed by incubation with 100fold molar excess of biotin-maleimide. Streptavidin and HER2 binding ofa biotinylated cys engineered ThioFab and a non-biotinylated wild typeFab was compared. The extent of biotin conjugation (interaction withstreptavidin) and their binding ability to HER2 were monitored by ELISAanalyses. Each Fab was tested at 2 ng and 20 ng.

Biotinylated A121C ThioFab retained comparable HER2 binding to that ofwild type hu4D5Fabv8 (FIG. 5). Wild type Fab and A121C-ThioFab werepurified by gel filtration column chromatography. The two samples weretested for HER2 and streptavidin binding by ELISA using goatanti-Fab-HRP as secondary antibody. Both wild type (open box) andThioFab (dotted box) have similar binding to HER2 but only ThioFabretained streptavidin binding. Only a background level of interactionwith streptavidin was observed with non-biotinylated wild typehu4D5Fabv8 (FIG. 5). Mass spectral (LC-ESI-MS) analysis ofbiotinylated-ThioFab (A121C) resulted in a major peak with 48294.5daltons compared to the wild type hu4D5Fabv8 (47737 daltons). The 537.5daltons difference between the two molecules exactly corresponds to asingle biotin-maleimide conjugated to the ThioFab. Mass spec proteinsequencing (LC-ESI-Tandem mass spec analysis) results further confirmedthat the conjugated biotin molecule was at the newly engineered Cysresidue (Table 4, Example 3).

Site Specific Conjugation of Biotin-maleimide to Albumin Binding Peptide(ABP)-thiofabs

Plasma-protein binding can be an effective means of improving thepharmacokinetic properties of short lived molecules. Albumin is the mostabundant protein in plasma. Serum albumin binding peptides (ABP) canalter the pharmacodynamics of fused active domain proteins, includingalteration of tissue uptake, penetration, and diffusion. Thesepharmacodynamic parameters can be modulated by specific selection of theappropriate serum albumin binding peptide sequence (US 20040001827). Aseries of albumin binding peptides were identified by phage displayscreening (Dennis et al. (2002) “Albumin Binding As A General StrategyFor Improving The Pharmacokinetics Of Proteins” J Biol. Chem.277:35035-35043; WO 01/45746). Compounds of the invention include ABPsequences taught by: (i) Dennis et al (2002) J Biol. Chem.277:35035-35043 at Tables III and IV, page 35038; (ii) US 20040001827 at[0076] SEQ ID NOS: 9-22; and (iii) WO 01/45746 at pages 12-13, SEQ IDNOS: z1-z14, and all of which are incorporated herein by reference.

Albumin Binding (ABP)-Fabs were engineered by fusing an albumin bindingpeptide to the C-terminus of Fab heavy chain in 1:1 stoichiometric ratio(1 ABP/1 Fab). It was shown that association of these ABP-Fabs withalbumin increased their half life by more than 25 fold in rabbits andmice. The above described reactive Cys residues can therefore beintroduced in these ABP-Fabs and used for site-specific conjugation withcytotoxic drugs followed by in vivo animal studies. FIG. 9 shows agraphical albumin binding peptide-Fab fusion (ABP-Fab) linker drugconjugate.

Exemplary albumin binding peptide sequences include, but are not limitedto the amino acid sequences listed in SEQ ID NOS: 1-5:

CDKTHTGGGSQRLMEDICLPRWGCLWEDDF SEQ ID NO: 1 QRLMEDICLPRWGCLWEDDF SEQ IDNO: 2 QRLIEDICLPRWGCLWEDDF SEQ ID NO: 3 RLIEDICLPRWGCLWEDD SEQ ID NO: 4DICLPRWGCLW SEQ ID NO: 5

The albumin binding peptide (ABP) sequences bind albumin from multiplespecies (mouse, rat, rabbit, bovine, rhesus, baboon, and human) with Kd(rabbit)=0.3 μM. The albumin binding peptide does not compete withligands known to bind albumin and has a half life (T½) in rabbit of 2.3hr. ABP-ThioFab proteins were purified on BSA-SEPHAROSE™ followed bybiotin-maleimide conjugation and purification on Superdex-S200 columnchromatography as described in previous sections. Purified biotinylatedproteins were homogeneous and devoid of any oligomeric forms (Example4).

FIG. 6 shows the properties of Albumin Binding Peptide (ABP)-ThioFabvariants. ELISA analyses were carried out to test the binding ability ofABP-hu4D5Fabv8-wt, ABP-hu4D5Fabv8-V110C and ABP-hu4D5Fabv8-A121C withrabbit albumin, streptavidin and HER2. Biotinylated ABP-ThioFabs arecapable of binding to albumin and HER2 with similar affinity to that ofwild type ABP-hu4D5Fabv8 as confirmed by ELISA (FIG. 6) and BIAcorebinding kinetics analysis (Table 3). An ELISA plate was coated withalbumin, HER2 and SA as described. Binding of biotinylated ABP-ThioFabsto albumin, HER2 and SA was probed with anti-Fab HRP. BiotinylatedABP-ThioFabs were capable of binding to streptavidin compared to nonbiotinylated control ABP-hu4D5Fabv8-wt indicating that ABP-ThioFabs wereconjugated with biotin maleimide like ThioFabs in a site specific manneras the same Cys mutants were used for both the variants (FIG. 6).

TABLE 3 BIAcore kinetic analysis for HER2 and rabbit albumin binding tobiotinylated ABP-hu4D5Fabv8 wild type and ThioFabs Antibody k_(on)(M⁻¹s⁻¹) k_(off) (s⁻¹) K_(d) (nM) HER2 binding wild type 4.57 × 10⁵ 4.19× 10⁻⁵ 0.0917 V110C 4.18 × 10⁵ 4.05 × 10⁻⁵ 0.097 A121C 3.91 × 10⁵ 4.15 ×10⁻⁵ 0.106 Rabbit albumin binding wild type 1.66 × 10⁵ 0.0206 124 V110C2.43 × 10⁵ 0.0331 136 A121C 1.70 × 10⁵ 0.0238 140 ABP = albumin bindingpeptide

Alternatively, an albumin-binding peptide may be linked to the antibodyby covalent attachment through a linker moiety.

Engineering of ABP-thiofabs with Two Free Thiol Groups per Fab

The above results indicate that all four (L-V15C, L-V110C, H-A88C andH-A121C) thioFab (cysteine engineered Fab antibodies) variants havereactive thiol groups that can be used for site specific conjugationwith a label reagent, linker reagent, or drug-linker intermediate.L-V15C can be expressed and purified but with relatively low yields.However the expression and purification yields of L-V110C, H-A88C andH-A121C variants were similar to that of hu4D5Fabv8. Therefore thesemutants can be used for further analysis and recombined to get more thanone thiol group per Fab. Towards this objective, one thiol group on thelight and one on heavy chain were constructed to obtain two thiol groupsper Fab molecule (L-V110C/H-A88C and L-V110C/H-A121C). These two doubleCys variants were expressed in an E. coli expression system andpurified. The homogeneity of purified biotinylated ABP-ThioFabs wasfound to be similar to that of single Cys variants.

The effects of engineering two reactive Cys residues per Fab wasinvestigated (FIG. 7). The presence of a second biotin was tested byprobing the binding of biotinylated ABP-ThioFab to SA usingstreptavidin-HRP (FIG. 7). For HER2/Fab analysis, an ELISA plate wascoated with HER2 and probed with anti-Fab HRP. For SA/Fab analysis, anELISA plate was coated with SA and probed with anti-Fab HRP. For SA/SAanalysis, an ELISA plate was coated with SA and probed with SA-HRP. FIG.7. ELISA analyses for the interaction of biotinylated ABP-hu4D5Fabv8 cysvariants with HER2, streptavidin (SA). HER2/Fab, SA/Fab and SA/SAindicate that their interactions were monitored by anti-Fab-HRP, SA-HRP,respectively. SA/Fab monitors the presence of single biotin per Fab andmore than one biotin per Fab is monitored by SA/SA analysis. Binding ofHER2 with double cys mutants is similar to that of single Cys variants(FIG. 7). However the extent of biotinylation on double Cys mutants washigher compared to single Cys variants due to more than one free thiolgroup per Fab molecule (FIG. 7).

Engineering of Thio IgG Variants of Trastuzumab

Cysteine was introduced into the full-length monoclonal antibody,trastuzumab (HERCEPTIN®, Genentech Inc.) at certain residues. The singlecys mutants H-A88C, H-A121C and L-V110C of trastuzumab, and double cysmutants V110C-A121C and V110C-A121C of trastuzumab were expressed in CHO(Chinese Hamster Ovary) cells by transient fermentation in mediacontaining 1 mM cysteine. The A88C mutant heavy chain sequence (450 aa)is SEQ ID NO:6. The A121C mutant heavy chain sequence (450 aa) is SEQ IDNO:7. The V110C mutant light chain sequence (214 aa) is SEQ ID NO:8.

SEQ ID NO: 6 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRCEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTCAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

According to one embodiment, the cysteine engineered thio-trastuzumabantibodies comprise one or more of the following variable region heavychain sequences with a free cysteine amino acid (SEQ ID NOS: 9-16).

Mutant Sequence SEQ ID NO: A40C WVRQCPGKGL SEQ ID NO: 9 A88C NSLRCEDTAVSEQ ID NO: 10 S119C LVTVCSASTKGPS SEQ ID NO: 11 S120C LVTVSCASTKGPS SEQID NO: 12 A121C LVTVSSCSTKGPS SEQ ID NO: 13 S122C LVTVSSACTKGPS SEQ IDNO: 14 A175C HTFPCVLQSSGLYS SEQ ID NO: 15 S179C HTFPAVLQCSGLYS SEQ IDNO: 16

According to another embodiment, the cysteine engineeredthio-trastuzumab antibodies comprise one or more of the followingvariable region light chain sequences with a free cysteine amino acid(SEQ ID NOS: 17-27).

Mutant Sequence SEQ ID NO: V15C SLSASCGDRVT SEQ ID NO: 17 A43CQKPGKCPKLLI SEQ ID NO: 18 V110C EIKRTCAAPSV SEQ ID NO: 19 S114CTCAAPCVFIFPP SEQ ID NO: 20 S121C FIFPPCDEQLK SEQ ID NO: 21 S127CDEQLKCGTASV SEQ ID NO: 22 A144C FYPRECKVQWK SEQ ID NO: 23 A153CWKVDNCLQSGN SEQ ID NO: 24 N158C ALQSGCSQESV SEQ ID NO: 25 S168CVTEQDCKDSTY SEQ ID NO: 26 V205C GLSSPCTKSFN SEQ ID NO: 27

The resulting full-length, thio-trastuzumab IgG variants were assayedfor thiol reactivity and HER2 binding activity. FIG. 13A shows a cartoondepiction of biotinylated antibody binding to immobilized HER2 and HRPlabeled secondary antibody for absorbance detection. FIG. 13B showsbinding measurements to immobilized HER2 with detection of absorbance at450 nm of (left to right): non-biotinylated wild type trastuzumab (Wt),biotin-maleimide conjugated thio-trastuzumab variants V110C (singlecys), A121C (single cys), and V110C-A121C (double cys). Each thio IgGvariant and trastuzumab was tested at 1, 10, and 100 ng. Themeasurements show that biotinylated anti-HER2ThioMabs retain HER2binding activity.

FIG. 14A shows a cartoon depiction of a biotinylated antibody binding toimmobilized HER2 with binding of biotin to anti-IgG-HRP for absorbancedetection. FIG. 14B shows binding measurements with detection ofabsorbance at 450 nm of biotin-maleimide conjugated thio-trastuzumabvariants and non-biotinylated wild type trastuzumab in binding tostreptavidin. From left to right: V110C (single cys), A121C (singlecys), V110C/A121C (double cys), and trastuzumab. Each thio IgGtrastuzumab variant and parent trastuzumab was tested at 1, 10, and 100ng. The measurements show that the HER2 ThioMabs have high thiolreactivity.

Cysteine was introduced into the full-length 2H9 anti-EphB2R antibody atcertain residues. The single cys mutant H-A121C of 2H9 was expressed inCHO (Chinese Hamster Ovary) cells by transient fermentation in mediacontaining 1 mM cysteine. The A121C₂H9 mutant heavy chain sequence (450aa) is SEQ ID NO:28.

SEQ ID NO: 28 EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWVGFINPSTGYTDYNQKFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRRPKIPRHANVFWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Cysteine engineered thio-2H9 antibodies comprise the following Fcconstant region heavy chain sequences with a free cysteine amino acid(SEQ ID NOS: 29-38).

Mutant Sequence SEQ ID NO: V273C HEDPECKFNWYVDGVEVHNAKTKPR SEQ ID NO: 29V279C HEDPEVKFNWYCDGVEVHNAKTKPR SEQ ID NO: 30 V282CHEDPEVKFNWYVDGCEVHNAKTKPR SEQ ID NO: 31 V284C HEDPEVKFNWYVDGVECHNAKTKPRSEQ ID NO: 32 A287C HEDPEVKFNWYVDGVEVHNCKTKPR SEQ ID NO: 33 S324CYKCKVCNKALP SEQ ID NO: 34 S337C IEKTICKAKGQPR SEQ ID NO: 35 A339CIEKTISKCKGQPR SEQ ID NO: 36 S375C KOFYPODIAVE SEQ ID NO: 37 S400CPPVLDCDGSFF SEQ ID NO: 38

FIG. 16 shows non-reducing (top) and reducing (bottom) denaturingSDS-PAGE (polyacrylamide gel electrophoresis) analysis of 2H9 ThioMab Fcvariants (left to right, lanes 1-9): A339C; S337C; S324C; A287C; V284C;V282C; V279C; and V273C, with 2H9 wild type, after purification onimmobilized Protein A. The lane on the right is a size marker ladder,indicating the intact proteins are about 150 kDa, heavy chain fragmentsabout 50 kDa, and light chain fragments about 25 kDa. FIG. 17A showsnon-reducing (left) and reducing (right) denaturing polyacrylamide gelelectrophoresis analysis of 2H9 ThioMab variants (left to right, lanes1-4): L-V15C; S179C; S375C; S400C, after purification on immobilizedProtein A. FIG. 17B shows non-reducing (left) and reducing (+DTT)(right) denaturing polyacrylamide gel electrophoresis analysis ofadditional 2H9 and 3A5 ThioMab variants after purification onimmobilized Protein A. The 2H9 ThioMab variants (in the Fab as well asFc region) were expressed and purified as described. As seen in FIGS.16, 17A and 17B, all the proteins are homogenous on SDS-PAGE followed bythe reduction and oxidation procedure of Example 11 to prepare reactiveThioMabs for conjugation (Example 12).

Cysteine was introduced into the full-length 3A5 anti-MUC16 antibody atcertain residues. The single cys mutant H-A121C of 3A5 was expressed inCHO (Chinese Hamster Ovary) cells by transient fermentation in mediacontaining 1 mM cysteine. The A121C 3A5 mutant heavy chain sequence (446aa) comprises SEQ ID NO:39.

SEQ ID NO: 39 DVQLQESGPGLVNPSQSLSLTCTVTGYSITNDYAWNWIRQFPGNKLEWMGYINYSGYTTYNPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCARWDGGLTYWGQGTLVTVSACSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Cysteine engineered thio-3A5 anti-MUC16 antibodies comprise thefollowing variable region heavy chain sequences with a free cysteineamino acid (SEQ ID NOS: 40-44).

Mutant Sequence SEQ ID NO: F45C NWIRQCPGNK SEQ ID NO: 40 A90CLNSCTTEDTAT SEQ ID NO: 41 A121C GQGTLVTVSACSTKGPSVFPL SEQ ID NO: 42A175C HTFPCVLQSSGLYS SEQ ID NO: 43 V176C HTFPACLQSSGLYS SEQ ID NO: 44

Cysteine engineered thio-3A5 anti-MUC16 antibodies comprise thefollowing variable region light chain sequences with a free cysteineamino acid (SEQ ID NOS: 45-49).

Mutant Sequence SEQ ID NO: L15C FLSVSCGGRVT SEQ ID NO: 45 A43CQKPGNCPRLLI SEQ ID NO: 46 V110C EIKRTCAAPSV SEQ ID NO: 47 A144CFYPRECKVQWK SEQ ID NO: 48 S168C VTEQDCKDSTY SEQ ID NO: 49

Thiol Reactivity of ThioMabs

The thiol reactivity of full length, IgG cysteine engineered antibodies(ThioMabs) was measured by biotinylation and streptavidin binding. Awestern blot assay was set up to screen the ThioMab that is specificallyconjugated with biotin-maleimide. In this assay, the antibodies areanalyzed on reducing SDS-PAGE and the presence of Biotin is specificallyprobed by incubating with streptavidin-HRP. As seen from FIG. 18, thestreptavidin-HRP interaction is either observed in heavy chain or lightchain depending on which engineered cys variant is being used and nointeraction is seen with wild type, indicating that ThioMab variantsspecifically conjugated the biotin at engineered Cys residue. FIG. 18shows denaturing gel analysis of reduced, biotinylated Thio-IgG variantsafter capture on immobilized anti-IgG-HRP (top gel) and streptavidin-HRP(bottom gel). Lane 1: 3A5H-A121C. Lane 2: 3A5 L-V1110C. Lane 3:2H9H-A121C. Lane 4: 2H9 L-V110C. Lane 5: anti-EphB2R 2H9 parent, wildtype. Each mutant (lanes 1-4) was captured by anti-IgG with HRPdetection (top) indicating that selectivity and affinity were retained.Capture by immobilized streptavidin with HRP detection (bottom)confirmed the location of biotin on heavy and light chains. The locationof cysteine mutation on the cysteine engineered antibodies in lanes 1and 3 is the heavy chain. The location of cysteine mutation on thecysteine engineered antibodies in lanes 2 and 4 is the light chain. Thecysteine mutation site undergoes conjugation with the biotin-maleimidereagent.

Analysis of the ThioMab cysteine engineered antibodies of FIG. 18 and a2H9 V15C variant by LC/MS gave quantitative indication of thiolreactivity (Table 5).

TABLE 5 LC/MS quantitation of biotinylation of ThioMabs - Thiolreactivity ThioMab variant number of biotin per ThioMab 2H9 wt 0.0 2H9L-V15C 0.6 2H9 L-V110C 0.5 2H9 H-A121C 2.0 3A5 L-V110C 1.0 3A5 H-A121C2.0

Cysteine engineering was conducted in the constant domain, i.e. Fcregion, of IgG antibodies. A variety of amino acid sites were convertedto cysteine sites and the expressed mutants, i.e. cysteine engineeredantibodies, were assessed for their thiol reactivity. Biotinylated 2H9ThioMab Fc variants were assessed for thiol reactivity by HRPquantitation by capture on immobilized streptavidin in an ELISA assay(FIG. 19). An ELISA assay was established to rapidly screen the Cysresidues with reactive Thiol groups. As depicted in FIG. 19 schematicdiagram, the streptavidin-biotin interaction is monitored by probingwith anti-IgG-HRP followed by measuring absorbance at 450 nm. Theseresults confirmed 2H9-ThioFc variants V282C, A287C, A339C, S375C andS400C had moderate to highest Thiol reactivity. The extent of biotinconjugation of 2H9 ThioMab Fc variants was quantitated by LS/MS analysisas reported in Table 6. The LS/MS analysis confirmed that the A282C,S375C and S400C variants had 100% biotin conjugation and V284C and A339Chad 50% conjugation, indicating the presence of a reactive cysteinethiol group. The other ThioFc variants, and the parent, wild type 2H9,had either very little biotinylation or none.

TABLE 6 LC/MS quantitation of biotinylation of 2H9 Fc ThioMabs 2H9ThioMab Fc variant % biotinylation V273C 0 V279C 31 V282C 100 V284C 50A287C 0 S324C 71 S337C 0 A339C 54 S375C 100 S400C 100 (wild type 2H9) 0

Thiol Reactivity of Thio-4D5 Fab Light Chain Variants

Screening of a variety of cysteine engineered light chain variant Fabsof the antiErbB2 antibody 4D5 gave a number of variants with a thiolreactivity value of 0.6 and higher (Table 7), as measured by thePHESELECTOR assay of FIG. 8. The thiol reactivity values of Table 7 arenormalized to the heavy chain 4D5 ThioFab variant (HC-A121C) which isset at 100%, assuming complete biotinylation of HC-A121C variant, andrepresented as percent values.

TABLE 7 Thiol reactivity percent values of 4D5 ThioFab light chainvariants Thiol reactivity value 4D5 ThioFab variant (%) V15C 100 V110C95 S114C 78 S121C 75 S127C 75 A153C 82 N158C 77 V205C 78 (HC-A121C) 100(4D5 wild type) 25

Antibody-drug Conjugates

The cysteine engineered antibodies of the invention may be conjugatedwith any therapeutic agent, i.e. drug moiety, which can be covalentlyattached to the antibody through a reactive cysteine thiol group.

An exemplary embodiment of an antibody-drug conjugate (ADC) compoundcomprises a cysteine engineered antibody (Ab), and a drug moiety (D)wherein the antibody has one or more free cysteine amino acids having athiol reactivity value in the range of 0.6 to 1.0, and the antibody isattached through the one or more free cysteine amino acids by a linkermoiety (L) to D; the composition having Formula I:

Ab-(L-D)_(p)  I

where p is 1, 2, 3, or 4. The number of drug moieties which may beconjugated via a thiol reactive linker moiety to an antibody molecule islimited by the number of cysteine residues which are introduced by themethods described herein. Exemplary ADC of Formula I therefore compriseantibodies which have 1, 2, 3, or 4 engineered cysteine amino acids.

Another exemplary embodiment of an antibody-drug conjugate compound(ADC) comprises a cysteine engineered antibody (Ab), an albumin-bindingpeptide (ABP) and a drug moiety (D) wherein the antibody is attached tothe drug moiety by a linker moiety (L) and the antibody is attached tothe albumin-binding peptide by an amide bond or a second linker moiety;the composition having Formula Ia:

ABP-Ab-(L-D)_(p)  Ia

where p is 1, 2, 3, or 4.

The ADC compounds of the invention include those with utility foranticancer activity. In particular, the compounds include acysteine-engineered antibody conjugated, i.e. covalently attached by alinker, to a drug moiety, i.e. toxin. When the drug is not conjugated toan antibody, the drug has a cytotoxic or cytostatic effect. Thebiological activity of the drug moiety is thus modulated by conjugationto an antibody. The antibody-drug conjugates (ADC) of the inventionselectively deliver an effective dose of a cytotoxic agent to tumortissue whereby greater selectivity, i.e. a lower efficacious dose, maybe achieved.

In one embodiment, the bioavailability of the ADC of the invention, oran intracellular metabolite of the ADC, is improved in a mammal whencompared to a drug compound comprising the drug moiety of the ADC. Also,the bioavailability of the ADC, or an intracellular metabolite of theADC is improved in a mammal when compared to the analog of the ADC nothaving the drug moiety.

Drug Moieties

The drug moiety (D) of the antibody-drug conjugates (ADC) includes anycompound, moiety or group which has a cytotoxic or cytostatic effect.Drug moieties include: (i) chemotherapeutic agents, which may functionas microtubulin inhibitors, mitosis inhibitors, topoisomeraseinhibitors, or DNA intercalators; (ii) protein toxins, which mayfunction enzymatically; and (iii) radioisotopes.

Exemplary drug moieties include, but are not limited to, a maytansinoid,an auristatin, a dolastatin, a trichothecene, CC1065, a calicheamicinand other enediyne antibiotics, a taxane, an anthracycline, andstereoisomers, isosteres, analogs or derivatives thereof.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PROC. NAT. ACAD. SCI. (USA)99:7968-7973), or maytansinol and maytansinol analogues preparedsynthetically according to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides). and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂₀R)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol). Many positions onmaytansine compounds are known to be useful as the linkage position,depending upon the type of link. For example, for forming an esterlinkage, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with a hydroxylgroup and the C-20 position having a hydroxyl group are all suitable.

The drug moiety (D) of the antibody-drug conjugates (ADC) of Formula Iinclude maytansinoids having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof D to a linker (L) of an antibody-drug conjugate (ADC). R mayindependently be H or a C₁-C₆ alkyl selected from methyl, ethyl,1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl,2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and3,3-dimethyl-2-butyl. The alkylene chain attaching the amide group tothe sulfur atom may be methanyl, ethanyl, or propyl, i.e. m is 1, 2, or3.

Maytansine compounds inhibit cell proliferation by inhibiting theformation of microtubules during mitosis through inhibition ofpolymerization of the microtubulin protein, tubulin (Remillard et al(1975) Science 189:1002-1005). Maytansine and maytansinoids are highlycytotoxic but their clinical use in cancer therapy has been greatlylimited by their severe systemic side-effects primarily attributed totheir poor selectivity for tumors. Clinical trials with maytansine hadbeen discontinued due to serious adverse effects on the central nervoussystem and gastrointestinal system (Issel et al (1978) Can. Treatment.Rev. 5:199-207).

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines (US 2005/0169933; WO 2005/037992; U.S. Pat. No.5,208,020).

As with other drug moieties, all stereoisomers of the maytansinoid drugmoiety are contemplated for the compounds of the invention, i.e. anycombination of R and S configurations at the chiral carbons of D. In oneembodiment, the maytansinoid drug moiety (D) will have the followingstereochemistry:

Exemplary embodiments of maytansinoid drug moieties include: DM1,(CR₂)_(m)═CH₂CH₂; DM3, (CR₂)_(m)═CH₂CH₂CH(CH₃); and DM4,(CR₂)_(m)═CH₂CH₂C(CH₃)₂, having the structures:

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

The drug moiety (D) of the antibody-drug conjugates (ADC) of Formula Ialso include dolastatins and their peptidic analogs and derivatives, theauristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). Various forms of adolastatin or auristatin drug moiety may be covalently attached to anantibody through the N (amino) terminus or the C (carboxyl) terminus ofthe peptidic drug moiety (WO 02/088172; Doronina et al (2003) NatureBiotechnology 21(7):778-784; Francisco et al (2003) Blood102(4):1458-1465).

Drug moieties include dolastatins, auristatins (U.S. Pat. No. 5,635,483;U.S. Pat. No. 5,780,588; U.S. Pat. No. 5,767,237; U.S. Pat. No.6,124,431), and analogs and derivatives thereof. Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in: WO2005/081711; Senter et al, Proceedings of the American Association forCancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004, the disclosure of each which are expressly incorporated byreference in their entirety.

The drug moiety (D) of the antibody-drug conjugates (ADC) of Formula Iinclude the monomethylauristatin drug moieties MMAE and MMAF linkedthrough the N-terminus to the antibody, and having the structures:

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry.

The drug moiety includes calicheamicin, and analogs and derivativesthereof. The calicheamicin family of antibiotics are capable ofproducing double-stranded DNA breaks at sub-picomolar concentrations.For the preparation of conjugates of the calicheamicin family, see U.S.Pat. No. 5,712,374; U.S. Pat. No. 5,714,586; U.S. Pat. No. 5,739,116;U.S. Pat. No. 5,767,285; U.S. Pat. No. 5,770,701, U.S. Pat. No.5,770,710; U.S. Pat. No. 5,773,001; U.S. Pat. No. 5,877,296. Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ₁^(I) (Hinman et al Cancer Research 53:3336-3342 (1993), Lode et alCancer Research 58:2925-2928 (1998).

Protein toxins include: diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain (Vitetta et al (1987) Science, 238:1098), abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes (WO 93/21232).

Therapeutic radioisotopes include: ³²P, ³³P, ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹³¹In,¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, ²¹²Pb, and radioactive isotopes ofLu.

The radioisotope or other labels may be incorporated in the conjugate inknown ways (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80:49-57; “Monoclonal ntibodies in Immunoscintigraphy” Chatal, CRC Press1989). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of a radionuclide to the antibody (WO 94/11026).

Linkers

A “Linker” (L) is a bifunctional or multifunctional moiety which can beused to link one or more Drug moieties (D) and an antibody unit (Ab) toform antibody-drug conjugates (ADC) of Formula I. Antibody-drugconjugates (ADC) can be conveniently prepared using a Linker havingreactive functionality for binding to the Drug and to the Antibody. Acysteine thiol of a cysteine engineered antibody (Ab) can form a bondwith a functional group of a linker reagent, a drug moiety ordrug-linker intermediate.

In one aspect, a Linker has a reactive site which has an electrophilicgroup that is reactive to a nucleophilic cysteine present on anantibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a Linker and forms a covalent bond to a Linker.Useful electrophilic groups include, but are not limited to, maleimideand haloacetamide groups.

Cysteine engineered antibodies react with linker reagents or drug-linkerintermediates, with electrophilic functional groups such as maleimide orα-halo carbonyl, according to the conjugation method at page 766 ofKlussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, andaccording to the protocol of Example 4.

In one embodiment, linker L of an ADC has the formula:

A_(a)-W_(w)-Y_(y)-

wherein:

-A- is a Stretcher unit covalently attached to a cysteine thiol of theantibody (Ab);

a is 0 or 1;

each —W— is independently an Amino Acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a Spacer unit covalently attached to the drug moiety; and

y is 0, 1 or 2.

Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking anantibody unit to an amino acid unit (—W—). In this regard an antibody(Ab) has a free cysteine thiol group that can form a bond with anelectrophilic functional group of a Stretcher Unit. RepresentativeStretcher units of this embodiment are depicted within the squarebrackets of Formulas IIIa and IIIb, wherein Ab-, —W—, —Y—, -D, w and yare as defined above, and R¹⁷ is a divalent radical selected from(CH₂)_(r), C₃-C₈ carbocyclyl, O—(CH₂)_(r), arylene, (CH₂)_(r)-arylene,-arylene-(CH₂)_(r)—, (CH₂)_(r)—(C₃-C₈ carbocyclyl), (C₃-C₈carbocyclyl)-(CH₂)_(r), C₃-C₈ heterocyclyl, (CH₂)_(r)—(C₃-C₈heterocyclyl), —(C₃-C₈ heterocyclyl)-(CH₂)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂)_(r)—, —(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—, and—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂)_(r)—; where R^(b) is H, C₁-C₆ alkyl,phenyl, or benzyl; and r is independently an integer ranging from 1-10.

Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbonatoms derived by the removal of two hydrogen atoms from a parentaromatic ring system. Typical arylene groups include, but are notlimited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

Heterocyclyl groups include a ring system in which one or more ringatoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system. Heterocycles are described in Paquette, Leo A.; “Principles ofModern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968),particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry ofHeterocyclic Compounds, A series of Monographs” (John Wiley & Sons, NewYork, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28;and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

Carbocyclyl groups include a saturated or unsaturated ring having 3 to 7carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples ofmonocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

It is to be understood from all the exemplary embodiments of Formula IADC such as III-VI, that even where not denoted expressly, from 1 to 4drug moieties are linked to an antibody (p=1-4), depending on the numberof engineered cysteine residues.

An illustrative Stretcher unit is that of Formula IIIa, and is derivedfrom maleimido-caproyl (MC) wherein R¹⁷ is —(CH₂)₅—:

An illustrative Stretcher unit is that of Formula IIIa, and is derivedfrom maleimido-propanoyl (MP) wherein R¹⁷ is —(CH₂)₂—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂— and r is 2:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— where R is H and each r is 2:

Another illustrative Stretcher unit is that of Formula IIIb wherein R¹⁷is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the Antibody unitvia a disulfide bond between a sulfur atom of the Antibody unit and asulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, Ab-, —W—, —Y—, -D, w and y are as defined above.

Ab-S—(—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(p)  IV

In yet another embodiment, the reactive group of the Stretcher containsa thiol-reactive functional group that can form a bond with a freecysteine thiol of an antibody. Examples of thiol-reaction functionalgroups include, but are not limited to, maleimide, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb, wherein —R¹⁷—, Ab-, —W—, —Y—,-D, w and y are as defined above;

In another embodiment, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) TetrahedronLetters 43:1987-1990). Dendritic linkers can increase the molar ratio ofdrug to antibody, i.e. loading, which is related to the potency of theADC. Thus, where a cysteine engineered antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Amino Acid Unit

The linker may comprise amino acid residues. The Amino Acid unit(—W_(w)—), when present, links the antibody (Ab) to the drug moiety (D)of the cysteine engineered antibody-drug conjugate (ADC) of theinvention.

—W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Amino acid residues which comprisethe Amino Acid unit include those occurring naturally, as well as minoramino acids and non-naturally occurring amino acid analogs, such ascitrulline. Each —W— unit independently has the formula denoted below inthe square brackets, and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug moiety (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D).

Useful —W_(w)— units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzymes, for example, atumor-associated protease. In one embodiment, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

Exemplary —W_(w)— Amino Acid units include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).

When R¹⁹ is other than hydrogen, the carbon atom to which R¹⁹ isattached is chiral. Each carbon atom to which R¹⁹ is attached isindependently in the (S) or (R) configuration, or a racemic mixture.Amino acid units may thus be enantiomerically pure, racemic, ordiastereomeric.

Spacer Unit

The Spacer unit (—Y—), when present (y=1 or 2), links an Amino Acid unit(—W_(w)—) to the drug moiety (D) when an Amino Acid unit is present(w=1-12). Alternately, the Spacer unit links the Stretcher unit to theDrug moiety when the Amino Acid unit is absent. The Spacer unit alsolinks the drug moiety to the antibody unit when both the Amino Acid unitand Stretcher unit are absent (w, y=0). Spacer units are of two generaltypes: self-immolative and non self-immolative. A non self-immolativeSpacer unit is one in which part or all of the Spacer unit remains boundto the Drug moiety after cleavage, particularly enzymatic, of an AminoAcid unit from the antibody-drug conjugate or the Drug moiety-linker.When an ADC containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved fromAb-A_(a)-W_(w)—. In one embodiment, an independent hydrolysis reactiontakes place within the target cell, cleaving the glycine-Drug moietybond and liberating the Drug.

In another embodiment, —Y_(y)— is a p-aminobenzylcarbamoyl (PAB) unit(see Schemes 2 and 3) whose phenylene portion is substituted with Q_(m)wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

Exemplary embodiments of a non self-immolative Spacer unit (—Y—) are:-Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one embodiment, a Drug moiety-linker or an ADC is provided in whichthe Spacer unit is absent (y=0), or a pharmaceutically acceptable saltor solvate thereof.

Alternatively, an ADC containing a self-immolative Spacer unit canrelease -D. In one embodiment, —Y— is a PAB group that is linked to—W_(w)— via the amino nitrogen atom of the PAB group, and connecteddirectly to -D via a carbonate, carbamate or ether group, where the ADChas the exemplary structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p ranges from 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al (1995) Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al(1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry, et al (1990) J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at glycine (Kingsbury et al(1984) J. Med. Chem. 27:1447) are also examples of self-immolativespacer useful in ADCs.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS), which can be used to incorporate andrelease multiple drugs, having the structure:

comprising a 2-(4-aminobenzylidene)propane-1,3-diol dendrimer unit (WO2004/043493; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494),wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges ranging from1 to 4.

Dendritic Linkers

In another embodiment, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11: 1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the ADC. Thus, where a cysteine engineeredantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker.

The following exemplary embodiments of dendritic linker reagents allowup to nine nucleophilic drug moiety reagents to be conjugated byreaction with the chloroethyl nitrogen mustard functional groups:

In another embodiment of a Spacer unit, branched, dendritic linkers withself-immolative 2,6-bis(hydroxymethyl)-p-cresol and2,4,6-tris(hydroxymethyl)-phenol dendrimer units (WO 2004/01993; Szalaiet al (2003) J. Amer. Chem. Soc. 125:15688-15689; Shamis et al (2004) J.Amer. Chem. Soc. 126:1726-1731; Amir et al (2003) Angew. Chem. Int. Ed.42:4494-4499) may be employed as linkers in the compounds of theinvention.

In another embodiment, the D moieties are the same.

In yet another embodiment, the D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4;

Embodiments of the Formula I antibody-drug conjugate compounds includeXIIIa (val-cit), XIIIb (MC-val-cit), XIIIc (MC-val-cit-PAB):

Other exemplary embodiments of the Formula Ia antibody-drug conjugatecompounds include XIVa-e:

where X is:

Y is:

and R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In another embodiment, a Linker has a reactive functional group whichhas a nucleophilic group that is reactive to an electrophilic grouppresent on an antibody. Useful electrophilic groups on an antibodyinclude, but are not limited to, aldehyde and ketone carbonyl groups.The heteroatom of a nucleophilic group of a Linker can react with anelectrophilic group on an antibody and form a covalent bond to anantibody unit. Useful nucleophilic groups on a Linker include, but arenot limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on anantibody provides a convenient site for attachment to a Linker.

Typically, peptide-type Linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press) which is well known in the field ofpeptide chemistry.

Linker intermediates may be assembled with any combination or sequenceof reactions including Spacer, Stretcher, and Amino Acid units. TheSpacer, Stretcher, and Amino Acid units may employ reactive functionalgroups which are electrophilic, nucleophilic, or free radical in nature.Reactive functional groups include, but are not limited to:

where X is a leaving group, e.g. O-mesyl, O-tosyl, —Cl, —Br, —I; ormaleimide.

In another embodiment, the Linker may be substituted with groups whichmodulated solubility or reactivity. For example, a charged substituentsuch as sulfonate (—SO₃ ⁻) or ammonium, may increase water solubility ofthe reagent and facilitate the coupling reaction of the linker reagentwith the antibody or the drug moiety, or facilitate the couplingreaction of Ab-L (antibody-linker intermediate) with D, or D-L(drug-linker intermediate) with Ab, depending on the synthetic routeemployed to prepare the ADC.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with linker reagents: BMPEO, BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), andincluding bis-maleimide reagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃,and BM(PEO)4, which are commercially available from PierceBiotechnology, Inc., Customer Service Department, P.O. Box 117,Rockford, Ill. 61105 U.S.A, U.S.A 1-800-874-3723, International+815-968-0747. See pages 467-498, 2003-2004 Applications Handbook andCatalog. Bis-maleimide reagents allow the attachment of the thiol groupof a cysteine engineered antibody to a thiol-containing drug moiety,label, or linker intermediate, in a sequential or concurrent fashion.Other functional groups besides maleimide, which are reactive with athiol group of a cysteine engineered antibody, drug moiety, label, orlinker intermediate include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

Useful linker reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem.60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; U.S.Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO03/026577; WO 03/043583; and WO 04/032828.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following linker reagents with the N-terminus of an Amino Acid unit:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of can be introduced into a Linker by reacting thefollowing bifunctional reagents with the N-terminus of an Amino Acidunit:

where X is Br or I. Stretcher units of formula can also be introducedinto a Linker by reacting the following bifunctional reagents with theN-terminus of an Amino Acid unit:

Stretcher units of formula (Va) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Isothiocyanate Stretchers of the formula shown below may be preparedfrom isothiocyanatocarboxylic acid chlorides as described in Angew.Chem., (1975) 87(14), 517.

wherein —R¹⁷— is as described herein.

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide Stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative Spacer has the structure:

where Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

An exemplary phe-lys(Mtr) dipeptide linker reagent having a maleimideStretcher unit and a p-aminobenzyl self-immolative Spacer unit can beprepared according to Dubowchik, et al. (1997) Tetrahedron Letters,38:5257-60, and has the structure:

where Mtr is mono-4-methoxytrityl, Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-halogen, -nitro or -cyano; and m is an integer ranging from 0-4.

Exemplary antibody-drug conjugate compounds of the invention include:

where Val is valine; Cit is citrulline; p is 1, 2, 3, or 4; and Ab is acysteine engineered antibody. Other exemplary antibody drug conjugateswhere maytansinoid drug moiety DM1 is linked through a BMPEO linker to athiol group of trastuzumab have the structure:

where Ab is a cysteine engineered antibody; n is 0, 1, or 2; and p is 1,2, 3, or 4.

Preparation of Antibody-drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a cysteine group of acysteine engineered antibody with a linker reagent, to formantibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a linker reagent, to formdrug-linker intermediate D-L, via a covalent bond, followed by reactionwith a cysteine group of a cysteine engineered antibody. Conjugationmethods (1) and (2) may be employed with a variety of cysteineengineered antibodies, drug moieties, and linkers to prepare theantibody-drug conjugates of Formula I.

Antibody cysteine thiol groups are nucleophilic and capable of reactingto form covalent bonds with electrophilic groups on linker reagents anddrug-linker intermediates including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides, such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups; and (iv) disulfides, including pyridyldisulfides, via sulfide exchange. Nucleophilic groups on a drug moietyinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

Maytansine may, for example, be converted to May-SSCH₃, which can bereduced to the free thiol, May-SH, and reacted with a modified antibody(Chari et al (1992) Cancer Research 52:127-131) to generate amaytansinoid-antibody immunoconjugate with a disulfide linker.Antibody-maytansinoid conjugates with disulfide linkers have beenreported (WO 04/016801; U.S. Pat. No. 6,884,874; US 2004/039176 A1; WO03/068144; US 2004/001838 A1; U.S. Pat. Nos. 6,441,163, 5,208,020,5,416,064; WO 01/024763). The disulfide linker SPP is constructed withlinker reagent N-succinimidyl 4-(2-pyridylthio) pentanoate.

Under certain conditions, the cysteine engineered antibodies may be madereactive for conjugation with linker reagents by treatment with areducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Full length,cysteine engineered monoclonal antibodies (ThioMabs) expressed in CHOcells were reduced with about a 50 fold excess of TCEP for 3 hrs at 37°C. to reduce disulfide bonds which may form between the newly introducedcysteine residues and the cysteine present in the culture media. Thereduced ThioMab was diluted and loaded onto HiTrap S column in 10 mMsodium acetate, pH 5, and eluted with PBS containing 0.3M sodiumchloride. Disulfide bonds were reestablished between cysteine residuespresent in the parent Mab with dilute (200 nM) aqueous copper sulfate(CuSO₄) at room temperature, overnight. Other oxidants, i.e. oxidizingagents, and oxidizing conditions, which are known in the art may beused. Ambient air oxidation is also effective. This mild, partialreoxidation step forms intrachain disulfides efficiently with highfidelity. An approximate 10 fold excess of drug-linker intermediate,e.g. BM(PEO)4-DM1 was added, mixed, and let stand for about an hour atroom temperature to effect conjugation and form the ThioMabantibody-drug conjugate. The conjugation mixture was gel filtered andloaded and eluted through a HiTrap S column to remove excess drug-linkerintermediate and other impurities.

FIG. 15 shows the general process to prepare a cysteine engineeredantibody expressed from cell culture for conjugation. Cysteine adducts,presumably along with various interchain disulfide bonds, arereductively cleaved to give a reduced form of the antibody. Theinterchain disulfide bonds between paired cysteine residues are reformedunder partial oxidation conditions, such as exposure to ambient oxygen.The newly introduced, engineered, and unpaired cysteine residues remainavailable for reaction with linker reagents or drug-linker intermediatesto form the antibody conjugates of the invention. The ThioMabs expressedin mammalian cell lines result in externally conjugated Cys adduct to anengineered Cys through —S—S— bond formation. Hence the purified ThioMabshave to be treated with reduction and oxidation procedures as describedin Example 11 to produce reactive ThioMabs. These ThioMabs are used toconjugate with maleimide containing cytotoxic drugs, fluorophores, andother labels.

A variety of ThioFab and ThioMab antibody-drug conjugates were prepared(Examples 4-8). Cysteine mutant hu4D5Fabv8 (V110C) was conjugated withthe maytansinoid drug moiety DM1 with a bis-maleimido linker reagentBMPEO to form hu4D5Fabv8 (V110C)-BMPEO-DM1 (Example 8).

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins, e.g. HER2, to the antibody of the ADC in a cell culturemedium; culturing the cells for a period from about 6 hours to about 5days; and measuring cell viability. Cell-based in vitro assays were usedto measure viability (proliferation), cytotoxicity, and induction ofapoptosis (caspase activation) of the ADC of the invention.

The in vitro potency of antibody-drug conjugates was measured by a cellproliferation assay (FIGS. 10 and 11, Example 9). The CellTiter-GloLuminescent Cell Viability Assay is a commercially available (PromegaCorp., Madison, Wis.), homogeneous assay method based on the recombinantexpression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713and 5,700,670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo Assay wasconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e. 3hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time. Alternatively, photonsfrom luminescence can be counted in a scintillation counter in thepresence of a scintillant. The light units can be represented then asCPS—counts per second.

The anti-proliferative effects of antibody-drug conjugates were measuredby the cell proliferation, in vitro cell killing assay above against theSK-BR-3 breast tumor cell line (FIGS. 10 and 11). IC₅₀ values of the ADCwere established against SK-BR-3 cells, which are known to overexpressHER2 receptor protein.

FIG. 10 shows that trastuzumab-SMCC-DM1 (IC₅₀=0.008-0.015 μg/ml) wasmore potent than the heavy chain cysteine mutant conjugatehu4D5Fabv8-(A121C)-BMPEO-DM1 (IC₅₀=0.04 μg/ml). Both conjugates weresignificantly more potent in cell killing than naked trastuzumab(IC₅₀=0.1 μg/ml). Drug loading for trastuzumab-SMCC-DM1 was 2.8 DM1/Aband for hu4D5Fabv8 (A121C)-BMPEO-DM1 was 0.6 DM1/Ab.

FIG. 11 shows that trastuzumab-SMCC-DM1 (IC₅₀=0.008-0.015 μg/ml) wasmore potent than the light chain cysteine mutant hu4D5Fabv8(V110C)-BMPEO-DM1 (IC₅₀=0.07 μg/ml). Both conjugates were more potent incell killing than naked trastuzumab (IC₅₀=0.1 μg/ml). Drug loading fortrastuzumab-SMCC-DM1 was 2.8 DM1/Ab and for hu4D5Fabv8 (V110C)-BMPEO-DM1was 0.9 DM1/Ab.

Full-length IgG ThioMab conjugates were tested for in vitro, cellproliferation efficacy and compared with parent antibodies. FIG. 20shows the results of an assay of SK-BR-3 cells treated with: parentantibody trastuzumab (HERCEPTIN®, Genentech, Inc.); trastuzumab-SMCC-DM1with a drug loading of about 3.4 DM1/Ab; and thio-trastuzumab(A121C)-BMPEO-DM1 with a drug loading of about 1.6 DM1/Ab. Thetrastuzumab-SMCC-DM1 conjugate is linked to the antibody via the aminoreactive, NHS ester SMCC linker reagent, whereas the thio-trastuzumab(A121C)-BMPEO-DM1 conjugates is linked via the thiol reactive, maleimideBMPEO linker reagent. Both conjugates were potent against SK-BR-3 cellsand showed comparable activity, whereas trastuzumab did not exert acytotoxic effect. FIG. 21A shows the results of an assay of HT 1080EphB2cells treated with: parent 2H9 anti-EphB2RX; and thio 2H9 (A121C)BMPEO-DM1 conjugate. FIG. 21B shows the results of an assay of BT 474cells treated with: parent 2H9 anti-EphB2RX; and thio 2H9 (A121C)BMPEO-DM1 conjugate. Against both HT 1080EphB2 and BT 474 cells, the 2H9ThioMab conjugate was more potent than the parent 2H9 antibodyconjugate. The conjugate Thio-2H9-BMPEO-DM1 showed functional cellkilling activity in EphB2 specific cell line (HT1080EphB2) compared to anon EphB2 cell line, BT474 in which only marginal activity is observed.

Antibody drug conjugates were compared where the antibody is a parentantibody and where the antibody is a cysteine engineered antibody. FIG.22 shows the results of an assay of PC3/neo cells treated with: 3A5 antiMUC16-SMCC-DM1; and thio 3A5 (A121C) BMPEO-DM1. FIG. 23 shows theresults of an assay of PC3/MUC16 cells treated with: 3A5 antiMUC16-SMCC-DM 1; and thio 3A5 (A121C) BMPEO-DM1. FIG. 24 shows theresults of an assay of OVCAR-3 cells treated with: 3A5 antiMUC16-SMCC-DM1; and thio 3A5 (A121C) BMPEO-DM1. Thio-3A5-BMPEO-DM1 didnot show any significant cell killing activity in the control PC3/neocell line, whereas it showed comparable activity to 3A5-SMCC-DM1 in thePC3/MUC16 cell line. Thio-3A5-DM1 conjugate also showed activity in theOVCAR-3 that expresses endogenous MUC16 antigen.

In Vivo Efficacy

The in vivo efficacy of two albumin binding peptide-DM1(maytansinoid)-antibody-drug conjugates (ADC) of the invention wasmeasured by a high expressing HER2 transgenic explant mouse model (FIG.12, Example 10). An allograft was propagated from the Fo5 mmtvtransgenic mouse which does not respond to, or responds poorly to,HERCEPTIN® therapy. Subjects were treated once withABP-rhuFab4D5-cys(light chain)-DM1; ABP-rhuFab4D5-cys(heavy chain)-DM1;and placebo PBS buffer control (Vehicle) and monitored over 3 weeks tomeasure the time to tumor doubling, log cell kill, and tumor shrinkage.

TDV Sample Dose Ti PR CR (days) Vehicle (PBS buffer) 7/7 0/7 0/7 3ABP-rhuFab4D5-   25 mg per kg 7/7 1/7 0/7 14 V110C (light chain)- (1012μg/m² of DM1) DM1 (0.9 DM1/Ab) ABP-rhuFab4D5- 37.5 mg per kg 7/7 4/7 0/716 A121C(heavy chain)- (1012 μg/m² of DM1) DM1 (0.6 DM1/Ab)

The term Ti is the number of animals in the study group with tumor atT═O— total animals in group. The term PR is the number of animalsattaining partial remission of tumor—animals with tumor at T=0÷in thegroup. The term CR is the number of animals attaining complete remissionof tumor÷animals with tumor at T=0 in the group. The term TDV is thetumor doubling time, i.e. time in days for the control tumor volume todouble.

The seven mice treated with 25 mg per kg (1012 ug/m² of DM1) ofABP-rhuFab4D5-cys(light chain)-DM 1 were all tumor-positive and gave oneanimal with partial remission after 20 days. The seven mice treated with37.5 mg per kg (1012 ug/m² of DM1) of ABP-rhuFab4D5-cys(heavy chain)-DM1were all tumor-positive and gave four animals with partial remissionafter 20 days.

The full length IgG ThioMab antibody variant with the A121C cysteinemutation and conjugated to the BMPEO linker and DM1 drug moiety wastested against the parent trastuzumab-SMCC-DM1 conjugate in MMTV-HER2Fo5 tumor-bearing mice. Tumor size at day 0 of injection was about100-200 mm in size. FIG. 25 shows the mean tumor volume change over 21days in athymic nude mice with MMTV-HER2 Fo5 mammary tumor allografts,after a single dose on Day 0 with: Vehicle (Buffer);trastuzumab-SMCC-DM110 mg/kg; thio trastuzumab (A121C)-SMCC-DM121 mg/kgand thio trastuzumab (A121C)-SMCC-DM1 10 mg/kg.

Sample Dose Ti PR CR Vehicle (PBS buffer) — 10/10 0/10 0/10trastuzumab-SMCC- 10 mg Ab per kg mouse 10/10 7/10 0/10 DM1 3.4 DM1/Ab(490 μg/m² of DM1) thio-trastuzumab 21 mg Ab per kg mouse  8/10 6/104/10 (A121C)-BMPEO-DM1 (496 μg/m² of DM1) 1.6 DM1/Ab thio-trastuzumab 10mg Ab per kg mouse 10/10 0/10 0/10 (A121C)-BMPEO-DM1 (236 μg/m² of DM1)1.6 DM1/Ab

It can be seen from FIG. 25 that each conjugate exerts a significanteffect of retarding tumor growth relative to placebo (Vehicle). Each ofthe ten mice in the four groups above received a single injection atday 1. The parent trastuzumab-SMCC-DM1 conjugate was loaded with morethan twice (3.4 DM1/Ab) the number of drug moieties than the cysteineengineered thio-trastuzumab (A121C)-BMPEO-DM1 conjugate (1.6 DM1/Ab).The effective amount of DM1 was thus approximately equal between parenttrastuzumab-SMCC-DM1 and the higher dose (21 mg Ab) thio-trastuzumab(A121C)-BMPEO-DM1. These two sample showed the most potency. After 14days post-injection, most of the animals receiving these conjugates werein partial or complete remission. The lower efficacy of the lower dosethio-trastuzumab (A121C)-BMPEO-DM1 sample confirmed a DM1 dose-relatedresponse. Thio-Trastuzumab-DM1 either dosed in equivalent antibody (10mg/kg) or DM1 drug (21 mg/kg) quantity to that of controltrastuzumab-SMCC-DM1 conjugate. As seen from the FIG. 25, Thio-BMPEO-DM1(21 mg/kg) showed slightly better response than that oftrastuzumab-SMCC-DM1 group as some of the animals showed completeresponse with Thiomab-DM 1 whereas there was only partial response withtrastuzumab-SMCC-DM 1.

Administration of Antibody-Drug Conjugates

The antibody-drug conjugates (ADC) of the invention may be administeredby any route appropriate to the condition to be treated. The ADC willtypically be administered parenterally, i.e. infusion, subcutaneous,intramuscular, intravenous, intradermal, intrathecal and epidural.

Pharmaceutical Formulations

Pharmaceutical formulations of therapeutic antibody-drug conjugates(ADC) of the invention are typically prepared for parenteraladministration, i.e. bolus, intravenous, intratumor injection with apharmaceutically acceptable parenteral vehicle and in a unit dosageinjectable form. An antibody-drug conjugate (ADC) having the desireddegree of purity is optionally mixed with pharmaceutically acceptablediluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation or an aqueous solution.

Acceptable diluents, carriers, excipients, and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Forexample, lyophilized anti-ErbB2 antibody formulations are described inWO 97/04801, expressly incorporated herein by reference.

The active pharmaceutical ingredients may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the ADC, which matrices are in the formof shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile,which is readily accomplished by filtration through sterile filtrationmembranes.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to 500 μg of the active ingredient per milliliter of solution inorder that infusion of a suitable volume at a rate of about 30 mL/hr canoccur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Although oral administration of protein therapeutics are disfavored dueto hydrolysis or denaturation in the gut, formulations of ADC suitablefor oral administration may be prepared as discrete units such ascapsules, cachets or tablets each containing a predetermined amount ofthe ADC.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Antibody-drug Conjugate Treatments

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia and lymphoid malignancies. Others include neuronal,glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial,stromal, blastocoelic, inflammatory, angiogenic and immunologic,including autoimmune, disorders.

The ADC compounds which are identified in the animal models andcell-based assays can be further tested in tumor-bearing higher primatesand human clinical trials. Human clinical trials can be designed similarto the clinical trials testing the efficacy of the anti-HER2 monoclonalantibody HERCEPTINO® in patients with HER2 overexpressing metastaticbreast cancers that had received extensive prior anti-cancer therapy asreported by Baselga et al. (1996) J. Clin. Oncol. 14:737-744. Theclinical trial may be designed to evaluate the efficacy of an ADC incombinations with known therapeutic regimens, such as radiation and/orchemotherapy involving known chemotherapeutic and/or cytotoxic agents.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

The cancer may comprise HER2-expressing cells, such that the ADC of thepresent invention are able to bind to the cancer cells. To determineErbB2 expression in the cancer, various diagnostic/prognostic assays areavailable. In one embodiment, ErbB2 overexpression may be analyzed byIHC, e.g. using the HERCEPTEST (Dako). Parrafin embedded tissue sectionsfrom a tumor biopsy may be subjected to the IHC assay and accorded aErbB2 protein staining intensity criteria as follows: Score 0, nostaining is observed or membrane staining is observed in less than 10%of tumor cells; Score 1+, a faint/barely perceptible membrane stainingis detected in more than 10% of the tumor cells, the cells are onlystained in part of their membrane; Score 2+, a weak to moderate completemembrane staining is observed in more than 10% of the tumor cells; Score3+, a moderate to strong complete membrane staining is observed in morethan 10% of the tumor cells. Those tumors with 0 or 1+scores for ErbB2overexpression assessment may be characterized as not overexpressingErbB2, whereas those tumors with 2+ or 3+ scores may be characterized asoverexpressing ErbB2.

Alternatively, or additionally, FISH assays such as the INFORM™ (VentanaCo., Ariz.) or PATHVISION™ (Vysis, Ill.) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of ErbB2 overexpression in the tumor.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupusnephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, for example, inflammatory bowel diseases (e.g., ulcerative colitisand Crohn's disease), autoimmune gastritis and pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, and celiac disease), vasculitis (such as, for example,ANCA-associated vasculitis, including Churg-Strauss vasculitis,Wegener's granulomatosis, and polyarteriitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of an anti-ErbB2 antibody.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays.

Combination Therapy

An antibody-drug conjugate (ADC) of the invention may be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen preferably has complementary activities to the ADC of thecombination such that they do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended. A pharmaceuticalcomposition containing an ADC of the invention may also have atherapeutically effective amount of a chemotherapeutic agent such as atubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

Other therapeutic regimens may be combined with the administration of ananticancer agent identified in accordance with this invention. Thecombination therapy may be administered as a simultaneous or sequentialregimen. When administered sequentially, the combination may beadministered in two or more administrations. The combined administrationincludes coadministration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.

In one embodiment, treatment with an ADC involves the combinedadministration of an anticancer agent identified herein, and one or morechemotherapeutic agents or growth inhibitory agents, includingcoadministration of cocktails of different chemotherapeutic agents.Chemotherapeutic agents include taxanes (such as paclitaxel anddocetaxel) and/or anthracycline antibiotics. Preparation and dosingschedules for such chemotherapeutic agents may be used according tomanufacturer's instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in “Chemotherapy Service”, (1992) Ed., M. C. Perry,Williams & Wilkins, Baltimore, Md.

The ADC may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (EP 616812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated ishormone independent cancer, the patient may previously have beensubjected to anti-hormonal therapy and, after the cancer becomes hormoneindependent, the ADC (and optionally other agents as described herein)may be administered to the patient. It may be beneficial to alsocoadminister a cardioprotectant (to prevent or reduce myocardialdysfunction associated with the therapy) or one or more cytokines to thepatient. In addition to the above therapeutic regimes, the patient maybe subjected to surgical removal of cancer cells and/or radiationtherapy.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g. ¹⁴C or ³H) ADC, administering it parenterally in adetectable dose (e.g. greater than about 0.5 mg/kg) to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur (typically about 30 seconds to 30 hours) andisolating its conversion products from the urine, blood or otherbiological samples. These products are easily isolated since they arelabeled (others are isolated by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, LC/MS or NMR analysis.In general, analysis of metabolites is done in the same way asconventional drug metabolism studies well-known to those skilled in theart. The conversion products, so long as they are not otherwise found invivo, are useful in diagnostic assays for therapeutic dosing of the ADCcompounds of the invention.

Labelled Antibody Imaging Methods

In another embodiment of the invention, cysteine engineered antibodiesmay be labelled through the cysteine thiol with radionuclides,fluorescent dyes, bioluminescence-triggering substrate moieties,chemiluminescence-triggering substrate moieties, enzymes, and otherdetection labels for imaging experiments with diagnostic,pharmacodynamic, and therapeutic applications. Generally, the labelledcysteine engineered antibody, i.e. “biomarker” or “probe”, isadministered by injection, perfusion, or oral ingestion to a livingorganism, e.g. human, rodent, or other small animal, a perfused organ,or tissue sample. The distribution of the probe is detected over a timecourse and represented by an image.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The containers may be formed from a varietyof materials such as glass or plastic. The container holds anantibody-drug conjugate (ADC) composition which is effective fortreating the condition and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). At least oneactive agent in the composition is an ADC. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES Example 1 Preparation of Biotinylated Thiofab Phage

ThioFab-phage (5×10¹² phage particles) were reacted with 150 fold excessof biotin-PEO-maleimide((+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctainediamine, Oda etal (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.)for 3 hours at room temperature. Excess biotin-PEO-maleimide was removedfrom biotin-conjugated phage by repeated PEG precipitations (3-4 times).Other commercially available biotinylation reagents with electrophilicgroups which are reactive with cysteine thiol groups may be used,including Biotin-BMCC, PEO-Iodoacetyl Biotin, Iodoacetyl-LC-Biotin, andBiotin-HPDP (Pierce Biotechnology, Inc.), andN^(α)-(3-maleimidylpropionyl)biocytin (MPB, Molecular Probes, Eugene,Oreg.). Other commercial sources for biotinylation, bifunctional andmultifunctional linker reagents include Molecular Probes, Eugene, Oreg.,and Sigma, St. Louis, Mo.

Example 2 PHESELECTOR Assay

Bovine serum albumin (BSA), erbB2 extracellular domain (HER2) andstreptavidin (100 μl of 2 μg/ml) were separately coated on Maxisorp 96well plates. After blocking with 0.5% Tween-20 (in PBS), biotinylatedand non-biotinylated hu4D5Fabv8-ThioFab-Phage (2×10¹⁰ phage particles)were incubated for 1 hour at room temperature followed by incubationwith horseradish peroxidase (HRP) labeled secondary antibody (anti-M13phage coat protein, pVIII protein antibody). FIG. 8 illustrates thePHESELECTOR Assay by a schematic representation depicting the binding ofFab or ThioFab to HER2 (top) and biotinylated ThioFab to streptavidin(bottom).

Standard HRP reaction was carried out and the absorbance was measured at450 nm. Thiol reactivity was measured by calculating the ratio betweenOD₄₅₀ for streptavidin/OD₄₅₀ for HER2. A thiol reactivity value of 1indicates complete biotinylation of the cysteine thiol. In the case ofFab protein binding measurements, hu4D5Fabv8 (2-20 ng) was used followedby incubation with HRP labeled goat polyclonal anti-Fab antibodies.

Example 3a Expression and Purification of ThioFabs

ThioFabs were expressed upon induction in 34B8, a non-suppressor E. colistrain (Baca et al (1997) Journal Biological Chemistry272(16):10678-84). The harvested cell pellet was resuspended in PBS(phosphate buffered saline), total cell lysis was performed by passingthrough a microfluidizer and the ThioFabs were purified by affinitychromatography with protein G SEPHAROSE™ (Amersham).

ThioFabs L-V15C, L-V110C, H-A88C, and H-A121C were expressed andpurified by Protein-G SEPHAROSE™ column chromatography. Oligomeric-Fabwas present in fractions 26 to 30, and most of the monomeric form was infractions 31-34. Fractions consisting of the monomeric form were pooledand analyzed by SDS-PAGE along with wild type hu4D5Fabv8 and analyzed onSDS-PAGE gel in reducing (with DTT or BME) and non-reducing (without DTTor BME) conditions. Gel filtration fractions of A121C-ThioFab wereanalyzed on non-reducing SDS-PAGE.

ThioFabs were conjugated with biotin-PEO-maleimide as described aboveand the biotinylated-ThioFabs were further purified by Superdex-200™(Amersham) gel filtration chromatography, which eliminated the freebiotin-PEO-maleimide and the oligomeric fraction of ThioFabs. Wild typehu4D5Fabv8 and hu4D5Fabv8 A121C-ThioFab (0.5 mg in quantity) were eachand separately incubated with 100 fold molar excess ofbiotin-PEO-maleimide for 3 hours at room temperature and loaded onto aSuperdex-200 gel filtration column to separate free biotin as well asoligomeric Fabs from the monomeric form.

Example 3b Analysis of ThioFabs

Enzymatic digest fragments of biotinylated hu4D5Fabv8 (A121C) ThioFaband wild type hu4D5Fabv8 were analyzed by liquid chromatographyelectrospray ionization mass spectroscopy (LS-ESI-MS) The differencebetween the 48294.5 primary mass of biotinylated hu4D5Fabv8 (A121C) andthe 47737.0 primary mass of wild type hu4D5Fabv8 was 557.5 mass units.This fragment indicates the presence of a single biotin-PEO-maleimidemoiety (C₂₃H₃₆N₅O₇S₂). Table 4 shows assignment of the fragmentationvalues which confirms the sequence.

TABLE 4 LC-ESI-Mass spec analysis of biotinylated hu4D5Fabv8 ThioFabA121C after tryptic digestion Amino acid b Fragment y Fragment A(Alanine) 72 M (Methionine) 203 2505 D (Aspartic acid) 318 2374 Y(Tyrosine) 481 2259 W (Tryptophan) 667 2096 G (Glycine) 724 1910 Q(glutamine) 852 1853 G (Glycine) 909 1725 T (Threonine) 1010 1668 L(Leucine) 1123 1567 V (Valine) 1222 1454 T (Threonine) 1323 1355 V(Valine) 1422 1254 S (Serine) 1509 1155 S (Serine) 1596 1068 C(Cysteine) + biotin 2242 981 S (Serine) 2329 335 T (Threonine) 2430 248K (Lysine) 175

Before and after Superdex-200 gel filtration, SDS-PAGE gel analyses,with and without reduction by DTT or BME, of biotinylatedABP-hu4D5Fabv8-A121C, biotinylated ABP— hu4D5Fabv8-V110C, biotinylateddouble Cys ABP-hu4D5Fabv8-(V110C-A88C), and biotinylated double CysABP-hu4D5Fabv8-(V110C-A121C) were conducted.

Mass spectroscopy analysis (MS/MS) of hu4D5Fabv8-(V110)-BMPEO-DM1 (afterSuperdex-200 gel filtration purification): Fab+1 51607.5, Fab 50515.5.This data shows 91.2% conjugation. MS/MS analysis ofhu4D5Fabv8-(V110C)-BMPEO-DM1 (reduced): LC 23447.2, LC+1 24537.3, HC(Fab) 27072.5. This data shows that all DM1 conjugation is on the lightchain of the Fab.

Example 4 Preparation of ABP-hu4D5Fabv8-(V110C)-MC-MMAE by Conjugationof ABP-hu4D5Fabv8-(V110C) and MC-MMAE

The drug linker reagent, maleimidocaproyl-monomethyl auristatin E(MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile andwater at known concentration, and added to chilledABP-hu4D5Fabv8-(V110C) ThioFab in phosphate buffered saline (PBS). Afterabout one hour, an excess of maleimide is added to quench the reactionand cap any unreacted antibody thiol groups. The reaction mixture isconcentrated by centrifugal ultrafiltration andABP-hu4D5Fabv8-(V110C)-MC-MMAE is purified and desalted by elutionthrough G25 resin in PBS, filtered through 0.2 μm filters under sterileconditions, and frozen for storage.

Example 5 Preparation of ABP-hu4D5Fabv8-(V110C)-MC-MMAF by conjugationof ABP-hu4D5Fabv8-(V110C) and MC-MMAF

ABP-hu4D5Fabv8-(V110C)-MC-MMAF is prepared by conjugation ofABP-hu4D5Fabv8-(V110C) ThioFab and MC-MMAF following the procedure ofExample 4.

Example 6 Preparation of ABP-A121C-ThioFab-MC-val-cit-PAB-MMAF byconjugation of ABP-A121C-ThioFab and MC-val-cit-PAB-MMAE

ABP-hu4D5Fabv8-(A121C)-MC-val-cit-PAB-MMAE is prepared by conjugation ofABP-hu4D5Fabv8-(A121C) and MC-val-cit-PAB-MMAE following the procedureof Example 4.

Example 7 Preparation of ABP-A121C-ThioFab-MC-val-cit-PAB-MMAF byconjugation of ABP-A121C-ThioFab and MC-val-cit-PAB-MMAFABP-hu4D5Fabv8-(A121C)-MC-val-cit-PAB-MMAF is prepared by conjugation ofABP-hu4D5Fabv8-(A121C) and MC-val-cit-PAB-MMAF following the procedureof Example 4.

Example 8 Preparation of hu4D5Fabv8-(V110C) ThioFab-BMPEO-DM1

The free cysteine on hu4D5Fabv8-(V110C) ThioFab was modified by thebis-maleimido reagent BM(PEO)4 (Pierce Chemical), leaving an unreactedmaleimido group on the surface of the antibody. This was accomplished bydissolving BM(PEO)4 in a 50% ethanol/water mixture to a concentration of10 mM and adding a tenfold molar excess of BM(PEO)4 to a solutioncontaining hu4D5Fabv8-(V110C) ThioFab in phosphate buffered saline at aconcentration of approximately 1.6 mg/ml (10 micromolar) and allowing itto react for 1 hour. Excess BM(PEO)4 was removed by gel filtration(HiTrap column, Pharmacia) in 30 mM citrate, pH 6 with 150 mM NaClbuffer. An approximate 10 fold molar excess DM1 dissolved in dimethylacetamide (DMA) was added to the hu4D5Fabv8-(V110C) ThioFab-BMPEOintermediate. Dimethylformamide (DMF) may also be employed to dissolvethe drug moiety reagent. The reaction mixture was allowed to reactovernight before gel filtration or dialysis into PBS to remove unreacteddrug. Gel filtration on S200 columns in PBS was used to remove highmolecular weight aggregates and furnish purified hu4D5Fabv8-(V110C)ThioFab-BMPEO-DM1.

By the same protocol, hu4D5Fabv8 (A121C) ThioFab-BMPEO-DM1 was prepared.

Example 9 In Vitro Cell Proliferation Assay

Efficacy of ADC were measured by a cell proliferation assay employingthe following protocol (CellTiter Glo Luminiscent Cell Viability Assay,Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res.62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells(SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each wellof a 96-well, opaque-walled plate.2. Control wells were prepared containing medium and without cells.3. ADC was added to the experimental wells and incubated for 3-5 days.4. The plates were equilibrated to room temperature for approximately 30minutes.5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well was added.6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Certain cells are seeded at 1000-2000/well (PC3 lines) or 2000-3000/well(OVCAR-3) in a 96-well plate, 50 uL/well. After one (PC3) or two(OVCAR-3) days, ADC are added in 50 μL volumes to final concentration of9000, 3000, 1000, 333, 111, 37, 12.4, 4.1, or 1.4 ng/mL, with “no ADC”control wells receiving medium alone. Conditions are in duplicate ortriplicate After 3 (PC3) or 4-5 (OVCAR-3) days, 100 μL/well CellTiterGlo II is added (luciferase-based assay; proliferation measured byATP levels) and cell counts are determined using a luminometer. Data areplotted as the mean of luminescence for each set of replicates, withstandard deviation error bars. The protocol is a modification of theCellTiter Glo Luminiscent Cell Viability Assay (Promega):

1. Plate 1000 cells/well of PC3/Muc16, PC3/neo (in 50 μL/well) of media.Ovcar3 cells should be plated at 2000 cells/well (in 50 μL) of theirmedia. (recipes below) Allow cells to attach overnight.

2. ADC is serially diluted 1:3 in media beginning at workingconcentration 18 μg/ml (this results in a final concentration of 9μg/ml). 50 μL of diluted ADC is added to the 50 μL of cells and mediaalready in the well.

3. Incubate 72-96 hrs (the standard is 72 hours, but watch the 0 ug/mLconcentration to stop assay when the cells are 85-95% confluent).

4. Add 100 μL/well of Promega Cell Titer Glo reagent, shake 3 min. andread on luminometer

Media: PC3/neo and PC3/MUC16 grow in 50/50/10% FBS/glutamine/250 μg/mLG-418 OVCAR-3 grow in RPMI/20% FBS/glutamine

Example 10 Tumor Growth Inhibition, in Vivo Efficacy in High ExpressingHER2 Transgenic Explant Mice

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Taconic (Germantown, N.Y.). Manystrains are suitable, but FVB female mice are preferred because of theirhigher susceptibility to tumor formation. FVB males were used for matingand vasectomized CD.1 studs were used to stimulate pseudopregnancy.Vasectomized mice can be obtained from any commercial supplier. Founderswere bred with either FVB mice or with 129/BL6xFVB p53 heterozygousmice. The mice with heterozygosity at p53 allele were used topotentially increase tumor formation. However, this has provenunnecessary. Therefore, some F1 tumors are of mixed strain. Foundertumors are FVB only. Six founders were obtained with some developingtumors without having litters.

Animals having tumors (allograft propagated from Fo5 mmtv transgenicmice) were treated with a single or multiple dose by IV injection ofADC. Tumor volume was assessed at various time points after injection.

Tumors arise readily in transgenic mice that express a mutationallyactivated form of neu, the rat homolog of HER2, but the HER2 that isoverexpressed in human breast cancers is not mutated and tumor formationis much less robust in transgenic mice that overexpress nonmutated HER2(Webster et al (1994) Semin. Cancer Biol. 5:69-76).

To improve tumor formation with nonmutated HER2, transgenic mice wereproduced using a HER2 cDNA plasmid in which an upstream ATG was deletedin order to prevent initiation of translation at such upstream ATGcodons, which would otherwise reduce the frequency of translationinitiation from the downstream authentic initiation codon of HER2 (forexample, see Child et al (1999) J. Biol. Chem. 274: 24335-24341).Additionally, a chimeric intron was added to the 5′ end, which shouldalso enhance the level of expression as reported earlier (Neuberger andWilliams (1988) Nucleic Acids Res. 16:6713; Buchman and Berg (1988) Mol.Cell. Biol. 8:4395; Brinster et al (1988) Proc. Natl. Acad. Sci. USA85:836). The chimeric intron was derived from a Promega vector, Pci-neomammalian expression vector (bp 890-1022). The cDNA 3′-end is flanked byhuman growth hormone exons 4 and 5, and polyadenylation sequences.Moreover, FVB mice were used because this strain is more susceptible totumor development. The promoter from MMTV-LTR was used to ensuretissue-specific HER2 expression in the mammary gland. Animals were fedthe AlN 76A diet in order to increase susceptibility to tumor formation(Rao et al (1997) Breast Cancer Res. and Treatment 45:149-158).

Example 11 Reduction/Oxidation of ThioMabs for Conjugation

Full length, cysteine engineered monoclonal antibodies (ThioMabs)expressed in CHO cells were reduced with about a 50 fold excess of TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) for 3 hrs at37° C. The reduced ThioMab (FIG. 15) was diluted and loaded onto aHiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBScontaining 0.3M sodium chloride. The eluted reduced ThioMab was treatedwith 200 nM aqueous copper sulfate (CuSO₄) at room temperature,overnight. Ambient air oxidation was also effective.

Example 12 Conjugation of ThioMabs

The reoxidized ThioMabs from Example 11, including thio-trastuzumab(A121C), thio-2H9 (A121C), and thio-3A5 (A121C), were combined with a 10fold excess of drug-linker intermediate, BM(PEO)4-DM1, mixed, and letstand for about an hour at room temperature to effect conjugation andform the ThioMab antibody-drug conjugates, including thio-trastuzumab(A121C)-BMPEO-DM1, thio-2H9 (A121C)-BMPEO-DM1, and thio-3A5(A121C)-BMPEO-DM1. The conjugation mixture was gel filtered, or loadedand eluted through a HiTrap S column to remove excess drug-linkerintermediate and other impurities.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

All patents, patent applications, and references cited throughout thespecification are expressly incorporated by reference.

1. A method of screening cysteine engineered antibodies for thiolreactivity comprising: (a) introducing one or more cysteine amino acidsinto a parent antibody in order to generate a cysteine engineeredantibody; (b) reacting the cysteine engineered antibody with athiol-reactive affinity reagent to generate an affinity labelled,cysteine engineered antibody; and (c) measuring the binding of theaffinity labelled, cysteine engineered antibody to a capture media. 2.The method of screening of claim 1 further comprising determining thethiol reactivity of the cysteine engineered antibody with thethiol-reactive reagent.
 3. The method of screening of claim 1 whereinstep (a) of the process comprises: (i) site-directed mutagenesis of agene encoding the cysteine engineered antibody in a double-stranded (ds)plasmid; and (ii) expressing the cysteine engineered antibody.
 4. Themethod of screening of claim 3 further comprising isolating andpurifying the expressed cysteine engineered antibody.
 5. The method ofscreening of claim 1 wherein the thiol-reactive affinity reagentcomprises a biotin moiety.
 6. The method of screening of claim 1 whereinthe thiol-reactive reagent comprises a maleimide moiety.
 7. The methodof screening of claim 1 wherein the capture media comprisesstreptavidin.
 8. The method of screening of claim 1 wherein the parentantibody is a fusion protein comprising an albumin-binding peptide(ABP).
 9. The method of screening of claim 8 wherein the ABP comprises asequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO:
 5. 10. The method of screening of claim 1 whereinthe parent antibody is an antibody fragment Fab.
 11. The method of claim1 wherein the cysteine engineered antibody is displayed on phage.